IGF-1R monoclonal antibodies and uses thereof

ABSTRACT

The present invention relates to conjugates including a chelating moiety of a metal complex thereof and a therapeutic or targeting moiety, methods for their production, and uses thereof.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in computer readable form. The computer readable form is incorporated herein by reference.

BACKGROUND

The insulin-like growth factor-1 receptor (IGF-1R) has been evaluated as a potential therapeutic target in the treatment of cancer. The present invention describes an alternate method for leveraging IGF-1R by delivering therapeutic radioisotopes that produce enhanced tumor efficacy with substantially lower doses than that of the antibody alone.

SUMMARY OF THE INVENTION

The present invention is directed to monoclonal antibodies that target the insulin-like growth factor-1 receptor and the radioimmunoconjugates thereof that demonstrate increased potency and enhance the excretion of a chelating moiety, or a metal complex thereof, when conjugated to a therapeutic moiety, a targeting moiety, or a cross-linking group.

Accordingly, in a first aspect, the invention features a compound having the structure: A-L¹-(L²)_(n)-B   Formula I

wherein A is chelating moiety or a metal complex thereof;

L¹ is optionally substituted C₁-C₆ alkyl, substituted C₁-C₆ heteroalkyl, substituted aryl or heteroaryl;

B is a therapeutic moiety, a targeting moiety, or cross-linking group,

or a pharmaceutically acceptable salt thereof;

n is 1-5;

each L², independently, has the structure: (-X¹-L³-Z¹-)   Formula II

wherein is X¹ is C═O(NR¹), C═S(NR¹), OC═O(NR¹), NR¹C═O(O), NR¹C═O(NR¹), —CH₂PhC═O(NR¹), —CH₂Ph(NH)C═S(NR¹), O, NR¹ and R¹ is H or optionally substituted C₁-C₆ alkyl or optionally substituted C₁-C₆ heteroalkyl, substituted aryl or heteroaryl; L³ is optionally substituted C₁-C₅₀ alkyl or optionally substituted C₁-C₅₀ heteroalkyl or C₅-C₂₀ polyethylene glycol; Z¹ is CH₂, C═O, C═S, OC═O, NR¹C═O, NR¹ and R¹ is a hydrogen or optionally substituted C₁-C₆ alkyl, pyrrolidine-2,5-dione.

In some embodiments, the chelating moiety is DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (1R,4R,7R,10R)-α, α′, α″, α′″-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra propionic acid), DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid), DOTA-GA anhydride (2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylene phosphonic acid)), DOTMP (1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylene phosphonic acid, DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonic acid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP (1,4,7-triazacyclononane-1,4,7-tri(methylene phosphonic acid), TETPA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetra acetic acid), HEHA (1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid), PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N′,N″,N′″, N″″-pentaacetic acid), H₄Octapa (N,N′-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N′-diacetic acid), H₂Dedpa (1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane), H₆phospa (N,N′-(methylenephosphonate)-N,N′-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane), TTHA (triethylenetetramine-N,N,N′,N″,N′″, N′″-hexaacetic acid), DO2P (tetraazacyclododecane dimethanephosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecanetriacetic acid), EDTA (ethylenediaminetetraacetic acid), Deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid-bismethylamide), HOPO (octadentate hydroxypyridinones) or porphyrin.

The person having ordinary skill in the art will understand that the use of chelating moieties in the practice of the invention are not limited to the specific constructs disclosed herein, but rather may include other known chelating moieties.

In some embodiments, the chelating moiety has the structure:

wherein Y¹ is —CH₂O CH₂(L²)_(n)-B, C═O(L²)_(n)-B, or C═S(L²)_(n)-B and Y² is —CH₂CO₂H;

wherein Y¹ is H, Y² is L¹-(L²)_(n)-B

In some embodiments, L¹ has the structure:

wherein R² is optionally substituted hydrogen or —CO₂H

In some embodiments, the metal can be selected from Bi, Pb, Y, Mn, Cr, Fe, Co, Zn, Ni, Tc, In, Ga, Cu, Re, Sm, a lanthanide, or an actinide, for use as imaging or therapeutic agents. Specific examples of radionuclides suitable for complexing to a compound of formula (I) include ⁴⁷Sc, ⁵⁵Co, ⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁸²Rb, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y, ⁸⁹Zr ⁹⁷Ru, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Pm, ¹⁴⁹Tb, ¹⁵³Sm, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁹Au, ²⁰¹Tl, ²⁰³Pb, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²⁵Ac, and ²²⁷Th.

In some embodiments, B is a therapeutic moiety or targeting moiety.

In some embodiments, the therapeutic moiety or targeting moiety is an antibody, an antigen-binding fragment thereof or other targeting protein such as nanobodies, affibodies, and consensus sequences from Fibronectin type III domains.

In some embodiments, the antibody, or an antigen-binding fragment thereof specifically binds insulin-like growth factor-1 receptor (IGF-1R), such as figitumumab, cixutumumab, ganitumab, AVE1642 (also known as humanized EM164 and huEM164), BIIB002, robatumumab, and teprotumumab. In some embodiments, the antibody, or an antigen binding fragment thereof is AVE1642 (SEQ ID NO: 9).

In some embodiments, the antibody, or antibody-binding fragment thereof includes a light chain variable domain including at least one, two, or all three complementarity determining regions (CDRs) selected from:

-   -   (a) CDR-L1 including the amino acid sequence of SEQ ID NO: 1;     -   (b) CDR-L2 including the amino acid sequence of SEQ ID NO: 2;         and     -   (c) CDR-L3 including the amino acid sequence of SEQ ID NO: 3.

In some embodiments, the antibody, or antibody-binding fragment thereof includes a heavy chain variable domain including at least one, two, or all three CDRs selected from:

(a) CDR-H1 including the amino acid sequence of SEQ ID NO: 5;

(b) CDR-H2 including the amino acid sequence of SEQ ID NO: 6; and

(c) CDR-H3 including the amino acid sequence of SEQ ID NO: 7.

In certain embodiments, the antibody, or antibody-binding fragment thereof includes a heavy chain variable domain and a light chain variable domain including at least one, two, three, four, five, or all six CDRs selected from:

(a) CDR-L1 including the amino acid sequence of SEQ ID NO: 1;

(b) CDR-L2 including the amino acid sequence of SEQ ID NO: 2;

(c) CDR-L3 including the amino acid sequence of SEQ ID NO: 3;

(d) CDR-H1 including the amino acid sequence of SEQ ID NO: 5;

(e) CDR-H2 including the amino acid sequence of SEQ ID NO: 6; and

(f) CDR-H3 including the amino acid sequence of SEQ ID NO: 7.

In other embodiments, the heavy chain variable domain includes the amino acid sequence of SEQ ID NO: 4.

In some embodiments, the light chain variable domain includes the amino acid sequence of SEQ ID NO: 8.

In some embodiments, the cross-linking group is an amino-reactive cross-linking group, a methionine-reactive cross-linking group, a thiol-reactive cross-linking group or a sortase-mediated coupling sequence.

In some embodiments, the amino-reactive, methionine-reactive, or thiol-reactive cross-linking group comprises an activated ester such as a hydroxysuccinimide ester, N-hydroxysulfosuccinimide, 2,3,5,6-tetrafluorophenol ester, 4-nitrophenol ester or an imidate, anhydride, thiol, disulfide, maleimide, azide, alkyne, strained alkyne, strained alkene, halogen, sulfonate, haloacetyl, amine, hydrazide, diazirine, phosphine, tetrazine, isothiocyanate, or an oxaziridine.

In some embodiments, the sortase recognition sequence may comprise of a terminal glycine-glycine-glycine (GGG) and/or LPTXG (SEQ ID NO: 10) amino acid sequence, where X is any amino acid.

The person having ordinary skill in the art will understand that the use of cross linking groups in the practice of the invention are not limited to the specific constructs disclosed herein, but rather may include other known cross linking groups.

In some embodiments, the cross-linking group is selected from the group consisting of:

In some embodiments, Y¹ is H.

In some embodiments, X¹ is C═O(NR¹) and R¹ is H.

In some embodiments, Z¹ is —CH₂.

In some embodiments, L² has n value of 1.

In some embodiments, the compound is selected from the group consisting of:

In some embodiments, the metal is a radionuclide.

In some embodiments, the radionuclide is ¹¹¹In.

In some embodiments, the radionuclide is ⁶⁸Ga.

In some embodiments, the radionuclide is ⁸⁶Y.

In some embodiments, the radionuclide is ⁸⁹Zr.

In some embodiments, the metal is a beta-emitting radionuclide.

In some embodiments, the radionuclide are ⁶⁷Cu, ¹⁷⁷Lu, or ⁹⁰Y

In some embodiments, the metal is an alpha-emitting radionuclide.

In some embodiments, the radionuclide is ²²⁵Ac, ²¹²Pb, ²²⁷Th or the progeny (daughter isotopes) thereof.

In another aspect, the invention feature a composition comprising any of the forgoing compounds and a metal salt. In some embodiments, the metal salt comprises a radionuclide (e.g., ¹¹¹In or ²²⁵Ac or the progeny thereof). In some embodiments, the metal salt is the nitrate salt, chloride salt, or acetate salt of ²²⁵Ac, or a combination of the chloride salt and acetate salt.

In another aspect, the invention features a pharmaceutical composition including any of the foregoing compounds and a pharmaceutically acceptable excipient.

In another aspect, the invention features a method of radiation treatment planning and/or radiation treatment, the method comprising administering to a subject in need thereof any of the foregoing compounds or pharmaceutical compositions.

In another aspect, the invention features a method of detecting and/or treating cancer, the method including administering to a subject in need thereof a first dose of any of the foregoing compounds or pharmaceutical compositions in an amount effective for radiation treatment planning, followed by administering subsequent doses of any of the foregoing compounds or pharmaceutical compositions in a therapeutically effective amount.

In some embodiments, the compound or composition administered in the first dose and the compound or composition administered in the second dose, or subsequent doses are the same.

In some embodiments, the compound or composition administered in the first dose and the compound or composition administered in the second dose, or subsequent doses are different.

In some embodiments, the cancer is a solid tumor or hematologic (liquid) cancer.

In some embodiments, the solid tumor cancer is breast cancer, non-small cell lung cancer, small cell lung cancer, pancreatic cancer, head and neck cancer, prostate cancer, colorectal cancer, sarcoma, adrenocortical carcinoma, neuroendocrine cancer, Ewing's Sarcoma, multiple myeloma, or acute myeloid leukemia.

In some embodiments, the foregoing methods further include administering an antiproliferative agent, radiation sensitizer, or an immunoregulatory or immunomodulatory agent.

In some embodiments, any of the foregoing compounds or compositions thereof and an antiproliferative agent or radiation sensitizer are administered within 28 days (e.g., within 14, 7, 6, 5, 4, 3, 2, or 1 day(s)) of each other.

In some embodiments, any of the above-described compounds or compositions thereof and an immunoregulatory or immunomodulatory agent are administered within 90 days (e.g., within 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 day(s)) of each other.

In another aspect, the invention features a method of making a radioconjugate (e.g., any of the radioconjugates described herein). The method includes the steps of (a) conjugating a bifunctional chelate to a biological molecule, (b) purifying the conjugate produced by step (a), and (c) chelating one or more radionuclides (e.g., one or more Ac-225 radionuclides) with the purified conjugate of step (b) at a temperature of less than 35° C. (e.g., 20-30° C.) to produce a radioconjugate (e.g. an actinium radioconjugate).

In some embodiments, the radioconjugate is a radioimmunoconjugate (e.g., any of the radioimmunoconjugates described herein).

In some embodiments, the pH of the reaction mixture of conjugation step (a) is 5.0-10.0 (e.g., 5.0-6.0, 6.0-7.0, 7.0-8.0, 8.0-9.0, or 9.0-10.0)

In some embodiments, the pH of the reaction mixture of conjugation step (a) is less than 6.4 (e.g., 6.3, 6.2, 6.1, 6.0, 5.9, or 5.8 or less).

In some embodiments, the pH of the reaction mixture of chelation step (c) is between 5.5 and 7.0 (e.g., 5.5-6.0, 6.0-6.5, or 6.5-7.0)

In some embodiments, the pH of the reaction mixture of chelating step (c) is less than 5.5 (e.g., 5.4, 5.3, 5.2, 5.1, or 5.0 or less) or more than 7.0 (e.g., 7.1, 7.2, 7.3, 7.4, 7.5 or more).

In some embodiments, the temperature of the reaction mixture of conjugation step (c) is 20-34° C. (e.g., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., or 34° C.).

Chemical Terms:

The term “acyl,” as used herein, represents a hydrogen or an alkyl group (e.g., a haloalkyl group), as defined herein, that is attached to the parent molecular group through a carbonyl group, as defined herein, and is exemplified by formyl (i.e., a carboxyaldehyde group), acetyl, trifluoroacetyl, propionyl, butanoyl and the like. Exemplary unsubstituted acyl groups include from 1 to 7, from 1 to 11, or from 1 to 21 carbons. In some embodiments, the alkyl group is further substituted with 1, 2, 3, or 4 substituents as described herein.

The term “alkyl,” as used herein, is inclusive of both straight chain and branched chain saturated groups from 1 to 20 carbons (e.g., from 1 to 10 or from 1 to 6), unless otherwise specified. Alkyl groups are exemplified by methyl, ethyl, n- and iso-propyl, n-, sec-, iso- and tert-butyl, neopentyl, and the like, and may be optionally substituted with one, two, three, or, in the case of alkyl groups of two carbons or more, four substituents independently selected from the group consisting of: (1) C₁₋₆ alkoxy; (2) C₁₋₆ alkylsulfinyl; (3) amino, as defined herein (e.g., unsubstituted amino (i.e., —NH₂) or a substituted amino (i.e., —N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8) hydroxy, optionally substituted with an O-protecting group; (9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇ spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO₂R^(A′), optionally substituted with an O-protecting group and where R^(A′) is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of —NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is, independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15) —C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently, selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′) is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) C₁₋₆ alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′), where each of R^(E′) and R^(F′) is, independently, selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of —NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is, independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19) —NR^(H′)C(O)R^(I′), wherein R^(H′) is selected from the group consisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I′) is selected from the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀ alkyl, and (h2) amino-polyethylene glycol of —NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is, independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20) —NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the group consisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selected from the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀ alkyl, and (h2) amino-polyethylene glycol of —NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is, independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21) amidine. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of a C₁-alkaryl can be further substituted with an oxo group to afford the respective aryloyl substituent.

The term “alkylene” and the prefix “alk-,” as used herein, represent a saturated divalent hydrocarbon group derived from a straight or branched chain saturated hydrocarbon by the removal of two hydrogen atoms, and is exemplified by methylene, ethylene, isopropylene, and the like. The term “C_(x-y) alkylene” and the prefix “C_(x-y) alk-” represent alkylene groups having between x and y carbons. Exemplary values for x are 1, 2, 3, 4, 5, and 6, and exemplary values for y are 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20 (e.g., C₁₋₆, C₁₋₁₀, C₂₋₂₀, C₂₋₆, C₂₋₁₀, or C₂₋₂₀ alkylene). In some embodiments, the alkylene can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein for an alkyl group.

The term “alkenyl,” as used herein, represents monovalent straight or branched chain groups of, unless otherwise specified, from 2 to 20 carbons (e.g., from 2 to 6 or from 2 to 10 carbons) containing one or more carbon-carbon double bonds and is exemplified by ethenyl, 1-propenyl, 2-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. Alkenyls include both cis and trans isomers. Alkenyl groups may be optionally substituted with 1, 2, 3, or 4 substituent groups that are selected, independently, from amino, aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl), as defined herein, or any of the exemplary alkyl substituent groups described herein.

The term “alkynyl,” as used herein, represents monovalent straight or branched chain groups from 2 to 20 carbon atoms (e.g., from 2 to 4, from 2 to 6, or from 2 to 10 carbons) containing a carbon-carbon triple bond and is exemplified by ethynyl, 1-propynyl, and the like. Alkynyl groups may be optionally substituted with 1, 2, 3, or 4 substituent groups that are selected, independently, from aryl, cycloalkyl, or heterocyclyl (e.g., heteroaryl), as defined herein, or any of the exemplary alkyl substituent groups described herein.

The term “amino,” as used herein, represents —N(R^(N1))₂, wherein each R^(N1) is, independently, H, OH, NO₂, N(R^(N2))₂, SO₂OR^(N2), SO₂R^(N2), SOR^(N2), an N-protecting group, alkyl, alkenyl, alkynyl, alkoxy, aryl, alkaryl, cycloalkyl, alkcycloalkyl, carboxyalkyl (e.g., optionally substituted with an O-protecting group, such as optionally substituted arylalkoxycarbonyl groups or any described herein), sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), alkoxycarbonylalkyl (e.g., optionally substituted with an O-protecting group, such as optionally substituted arylalkoxycarbonyl groups or any described herein), heterocyclyl (e.g., heteroaryl), or alkheterocyclyl (e.g., alkheteroaryl), wherein each of these recited R^(N1) groups can be optionally substituted, as defined herein for each group; or two R^(N1) combine to form a heterocyclyl or an N-protecting group, and wherein each R^(N2) is, independently, H, alkyl, or aryl. The amino groups of the invention can be an unsubstituted amino (i.e., —NH₂) or a substituted amino (i.e., —N(R^(N1))₂). In a preferred embodiment, amino is —NH₂ or —NHR^(N1), wherein R^(N1) is, independently, OH, NO₂, NH₂, NR^(N2) ₂, SO₂OR^(N2), SO₂R^(N2), SOR^(N2), alkyl, carboxyalkyl, sulfoalkyl, acyl (e.g., acetyl, trifluoroacetyl, or others described herein), alkoxycarbonylalkyl (e.g., t-butoxycarbonylalkyl) or aryl, and each R^(N2) can be H, C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), or C₆₋₁₀ aryl.

The term “amino acid,” as described herein, refers to a molecule having a side chain, an amino group, and an acid group (e.g., a carboxy group of —CO₂H or a sulfo group of —SO₃H), wherein the amino acid is attached to the parent molecular group by the side chain, amino group, or acid group (e.g., the side chain). In some embodiments, the amino acid is attached to the parent molecular group by a carbonyl group, where the side chain or amino group is attached to the carbonyl group. Exemplary side chains include an optionally substituted alkyl, aryl, heterocyclyl, alkaryl, alkheterocyclyl, aminoalkyl, carbamoylalkyl, and carboxyalkyl. Exemplary amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, hydroxynorvaline, isoleucine, leucine, lysine, methionine, norvaline, ornithine, phenylalanine, proline, pyrrolysine, selenocysteine, serine, taurine, threonine, tryptophan, tyrosine, and valine. Amino acid groups may be optionally substituted with one, two, three, or, in the case of amino acid groups of two carbons or more, four substituents independently selected from the group consisting of: (1) C₁₋₆ alkoxy; (2) C₁₋₆ alkylsulfinyl; (3) amino, as defined herein (e.g., unsubstituted amino (i.e., —NH₂) or a substituted amino (i.e., —N(R^(N1))₂, where R^(N1) is as defined for amino); (4) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (5) azido; (6) halo; (7) (C₂₋₉ heterocyclyl)oxy; (8) hydroxy; (9) nitro; (10) oxo (e.g., carboxyaldehyde or acyl); (11) C₁₋₇ spirocyclyl; (12) thioalkoxy; (13) thiol; (14) —CO₂R^(A′), where R^(A′) is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of —NR^(N′)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is, independently, hydrogen or optionally substituted C₁₋₆ alkyl; (15) —C(O)NR^(B′)R^(C′), where each of R^(B′) and R^(C′) is, independently, selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (16) —SO₂R^(D′), where R^(D′) is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) 01-6 alk-C₆₋₁₀ aryl, and (d) hydroxy; (17) —SO₂NR^(E′)R^(F′), where each of R^(E′) and R^(F′) is, independently, selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (18) —C(O)R^(G′), where R^(G′) is selected from the group consisting of (a) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c) C₆₋₁₀ aryl, (d) hydrogen, (e) C₁₋₆ alk-C₆₋₁₀ aryl, (f) amino-C₁₋₂₀ alkyl, (g) polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀ alkyl, and (h) amino-polyethylene glycol of —NR^(N1) (CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is, independently, hydrogen or optionally substituted C₁₋₆ alkyl; (19) —NR^(H′)C(O)R^(I′), wherein R^(H′) is selected from the group consisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(I′) is selected from the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀ alkyl, and (h2) amino-polyethylene glycol of —NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is, independently, hydrogen or optionally substituted C₁₋₆ alkyl; (20) —NR^(J′)C(O)OR^(K′), wherein R^(J′) is selected from the group consisting of (a1) hydrogen and (b1) C₁₋₆ alkyl, and R^(K′) is selected from the group consisting of (a2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl), (b2) C₂₋₂₀ alkenyl (e.g., C₂₋₆ alkenyl), (c2) C₆₋₁₀ aryl, (d2) hydrogen, (e2) C₁₋₆ alk-C₆₋₁₀ aryl, (f2) amino-C₁₋₂₀ alkyl, (g2) polyethylene glycol of —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)OR′, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and R′ is H or C₁₋₂₀ alkyl, and (h2) amino-polyethylene glycol of —NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1), wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is, independently, hydrogen or optionally substituted C₁₋₆ alkyl; and (21) amidine. In some embodiments, each of these groups can be further substituted as described herein.

The term “aryl,” as used herein, represents a mono-, bicyclic, or multicyclic carbocyclic ring system having one or two aromatic rings and is exemplified by phenyl, naphthyl, 1,2-dihydronaphthyl, 1,2,3,4-tetrahydronaphthyl, anthracenyl, phenanthrenyl, fluorenyl, indanyl, indenyl, and the like, and may be optionally substituted with 1, 2, 3, 4, or 5 substituents independently selected from the group 5 consisting of: (1) C₁₋₇ acyl (e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g., perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such as perfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q is an integer from zero to four, and R^(A′) is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to four and where R^(B′) and R^(C′) are independently selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q is an integer from zero to four and where R^(D′) is selected from the group consisting of (a) alkyl, (b) C₆₋₁₀ aryl, and (c) alk-C₆₋₁₀ aryl; (20) —(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four and where each of R^(E′) and R^(F′) is, independently, selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈ cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂ heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) C₂₋₂₀ alkenyl; and (27) C₂₋₂₀ alkynyl. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of a C₁-alkaryl or a C₁-alkheterocyclyl can be further substituted with an oxo group to afford the respective aryloyl and (heterocyclyl)oyl substituent group.

The term “arylalkyl,” as used herein, represents an aryl group, as defined herein, attached to the parent molecular group through an alkylene group, as defined herein. Exemplary unsubstituted arylalkyl groups are from 7 to 30 carbons (e.g., from 7 to 16 or from 7 to 20 carbons, such as C₁₋₆ alk-C₆₋₁₀-aryl, C₁₋₁₀ alk-C₆₋₁₀ aryl, or C₁₋₂₀ alk-C₆₋₁₀ aryl). In some embodiments, the alkylene and the aryl each can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein for the respective groups. Other groups preceded by the prefix “alk-” are defined in the same manner, where “alk” refers to a C₁₋₆ alkylene, unless otherwise noted, and the attached chemical structure is as defined herein.

The term “carbonyl,” as used herein, represents a C(O) group, which can also be represented as C═O.

The term “carboxy,” as used herein, means —CO₂H.

The term “cyano,” as used herein, represents an —CN group.

The term “cycloalkyl,” as used herein represents a monovalent saturated or unsaturated non-aromatic cyclic hydrocarbon group from three to eight carbons, unless otherwise specified, and is exemplified by cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicycle heptyl, and the like. When the cycloalkyl group includes one carbon-carbon double bond or one carbon-carbon triple bond, the cycloalkyl group can be referred to as a “cycloalkenyl” or “cycloalkynyl” group respectively. Exemplary cycloalkenyl and cycloalkynyl groups include cyclopentenyl, cyclohexenyl, cyclohexynyl, and the like. The cycloalkyl groups of this invention can be optionally substituted with: (1) C₁₋₇ acyl (e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g., perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such as perfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g., C₁₋₁₂ heteroaryl); (13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q is an integer from zero to four, and R^(A′) is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to four and where R^(B′) and R^(C′) are independently selected from the group consisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q is an integer from zero to four and where R^(D′) is selected from the group consisting of (a) C₆₋₁₀ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀ aryl; (20) —(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four and where each of R^(E′) and R^(F′) is, independently, selected from the group consisting of (a) hydrogen, (b) C₆₋₁₀ alkyl, (c) C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈ cycloalkoxy; (24) C₆₋₁₀ aryl-C₁₋₆ alkoxy; (25) C₁₋₆ alk-C₁₋₁₂ heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) C₂₋₂₀ alkenyl; and (28) C₂₋₂₀ alkynyl. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of a C₁-alkaryl or a C₁-alkheterocyclyl can be further substituted with an oxo group to afford the respective aryloyl and (heterocyclyl)oyl substituent group.

The term “diastereomer,” as used herein means stereoisomers that are not mirror images of one another and are non-superimposable on one another.

The term “enantiomer,” as used herein, means each individual optically active form of a compound of the invention, having an optical purity or enantiomeric excess (as determined by methods standard in the art) of at least 80% (i.e., at least 90% of one enantiomer and at most 10% of the other enantiomer), preferably at least 90% and more preferably at least 98%.

The term “halogen,” as used herein, represents a halogen selected from bromine, chlorine, iodine, or fluorine.

The term “heteroalkyl,” as used herein, refers to an alkyl group, as defined herein, in which one or two of the constituent carbon atoms have each been replaced by nitrogen, oxygen, or sulfur. In some embodiments, the heteroalkyl group can be further substituted with 1, 2, 3, or 4 substituent groups as described herein for alkyl groups. The terms “heteroalkenyl” and heteroalkynyl,” as used herein refer to alkenyl and alkynyl groups, as defined herein, respectively, in which one or two of the constituent carbon atoms have each been replaced by nitrogen, oxygen, or sulfur. In some embodiments, the heteroalkenyl and heteroalkynyl groups can be further substituted with 1, 2, 3, or 4 substituent groups as described herein for alkyl groups.

The term “heteroaryl,” as used herein, represents that subset of heterocyclyls, as defined herein, which are aromatic: i.e., they contain 4n+2 pi electrons within the mono- or multicyclic ring system. Exemplary unsubstituted heteroaryl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. In some embodiment, the heteroaryl is substituted with 1, 2, 3, or 4 substituents groups as defined for a heterocyclyl group.

The term “heteroarylalkyl” refers to a heteroaryl group, as defined herein, attached to the parent molecular group through an alkylene group, as defined herein. Exemplary unsubstituted heteroarylalkyl groups are from 2 to 32 carbons (e.g., from 2 to 22, from 2 to 18, from 2 to 17, from 2 to 16, from 3 to 15, from 2 to 14, from 2 to 13, or from 2 to 12 carbons, such as C₁₋₆ alk-C₁₋₁₂ heteroaryl, C₁₋₁₀ alk-C₁₋₁₂ heteroaryl, or C₁₋₂₀ alk-C₁₋₁₂ heteroaryl). In some embodiments, the alkylene and the heteroaryl each can be further substituted with 1, 2, 3, or 4 substituent groups as defined herein for the respective group. Heteroarylalkyl groups are a subset of heterocyclylalkyl groups.

The term “heterocyclyl,” as used herein represents a 5-, 6- or 7-membered ring, unless otherwise specified, containing one, two, three, or four heteroatoms independently selected from the group consisting of nitrogen, oxygen, and sulfur. The 5-membered ring has zero to two double bonds, and the 6- and 7-membered rings have zero to three double bonds. Exemplary unsubstituted heterocyclyl groups are of 1 to 12 (e.g., 1 to 11, 1 to 10, 1 to 9, 2 to 12, 2 to 11, 2 to 10, or 2 to 9) carbons. The term “heterocyclyl” also represents a heterocyclic compound having a bridged multicyclic structure in which one or more carbons and/or heteroatoms bridges two non-adjacent members of a monocyclic ring, e.g., a quinuclidinyl group. The term “heterocyclyl” includes bicyclic, tricyclic, and tetracyclic groups in which any of the above heterocyclic rings is fused to one, two, or three carbocyclic rings, e.g., an aryl ring, a cyclohexane ring, a cyclohexene ring, a cyclopentane ring, a cyclopentene ring, or another monocyclic heterocyclic ring, such as indolyl, quinolyl, isoquinolyl, tetrahydroquinolyl, benzofuryl, benzothienyl and the like. Examples of fused heterocyclyls include tropanes and 1,2,3,5,8,8a-hexahydroindolizine. Heterocyclics include pyrrolyl, pyrrolinyl, pyrrolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyridyl, piperidinyl, homopiperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, pyridazinyl, oxazolyl, oxazolidinyl, isoxazolyl, isoxazolidiniyl, morpholinyl, thiomorpholinyl, thiazolyl, thiazolidinyl, isothiazolyl, isothiazolidinyl, indolyl, indazolyl, quinolyl, isoquinolyl, quinoxalinyl, dihydroquinoxalinyl, quinazolinyl, cinnolinyl, phthalazinyl, benzimidazolyl, benzothiazolyl, benzoxazolyl, benzothiadiazolyl, furyl, thienyl, thiazolidinyl, isothiazolyl, triazolyl, tetrazolyl, oxadiazolyl (e.g., 1,2,3-oxadiazolyl), purinyl, thiadiazolyl (e.g., 1,2,3-thiadiazolyl), tetrahydrofuranyl, dihydrofuranyl, tetrahydrothienyl, dihydrothienyl, dihydroindolyl, dihydroquinolyl, tetrahydroquinolyl, tetrahydroisoquinolyl, dihydroisoquinolyl, pyranyl, dihydropyranyl, dithiazolyl, benzofuranyl, isobenzofuranyl, benzothienyl, and the like, including dihydro and tetrahydro forms thereof, where one or more double bonds are reduced and replaced with hydrogens. Still other exemplary heterocyclyls include: 2,3,4,5-tetrahydro-2-oxo-oxazolyl; 2,3-dihydro-2-oxo-1H-imidazolyl; 2,3,4,5-tetrahydro-5-oxo-1H-pyrazolyl (e.g., 2,3,4,5-tetrahydro-2-phenyl-5-oxo-1H-pyrazolyl); 2,3,4,5-tetrahydro-2,4-dioxo-1H-imidazolyl (e.g., 2,3,4,5-tetrahydro-2,4-dioxo-5-methyl-5-phenyl-1H-imidazolyl); 2,3-dihydro-2-thioxo-1,3,4-oxadiazolyl (e.g., 2,3-dihydro-2-thioxo-5-phenyl-1,3,4-oxadiazolyl); 4,5-dihydro-5-oxo-1H-triazolyl (e.g., 4,5-dihydro-3-methyl-4-amino 5-oxo-1H-triazolyl); 1,2,3,4-tetrahydro-2,4-dioxopyridinyl (e.g., 1,2,3,4-tetrahydro-2,4-dioxo-3,3-diethylpyridinyl); 2,6-dioxo-piperidinyl (e.g., 2,6-dioxo-3-ethyl-3-phenylpiperidinyl); 1,6-dihydro-6-oxopyridiminyl; 1,6-dihydro-4-oxopyrimidinyl (e.g., 2-(methylthio)-1,6-dihydro-4-oxo-5-methylpyrimidin-1-yl); 1,2,3,4-tetrahydro-2,4-dioxopyrimidinyl (e.g., 1,2,3,4-tetrahydro-2,4-dioxo-3-ethylpyrimidinyl); 1,6-dihydro-6-oxo-pyridazinyl (e.g., 1,6-dihydro-6-oxo-3-ethylpyridazinyl); 1,6-dihydro-6-oxo-1,2,4-triazinyl (e.g., 1,6-dihydro-5-isopropyl-6-oxo-1,2,4-triazinyl); 2,3-dihydro-2-oxo-1H-indolyl (e.g., 3,3-dimethyl-2,3-dihydro-2-oxo-1H-indolyl and 2,3-dihydro-2-oxo-3,3′-spiropropane-1H-indol-1-yl); 1,3-dihydro-1-oxo-2H-iso-indolyl; 1,3-dihydro-1,3-dioxo-2H-iso-indolyl; 1H-benzopyrazolyl (e.g., 1-(ethoxycarbonyl)-1H-benzopyrazolyl); 2,3-dihydro-2-oxo-1H-benzimidazolyl (e.g., 3-ethyl-2,3-dihydro-2-oxo-1H-benzimidazolyl); 2,3-dihydro-2-oxo-benzoxazolyl (e.g., 5-chloro-2,3-dihydro-2-oxo-benzoxazolyl); 2,3-dihydro-2-oxo-benzoxazolyl; 2-oxo-2H-benzopyranyl; 1,4-benzodioxanyl; 1,3-benzodioxanyl; 2,3-dihydro-3-oxo,4H-1,3-benzothiazinyl; 3,4-dihydro-4-oxo-3H-quinazolinyl (e.g., 2-methyl-3,4-dihydro-4-oxo-3H-quinazolinyl); 1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl (e.g., 1-ethyl-1,2,3,4-tetrahydro-2,4-dioxo-3H-quinazolyl); 1,2,3,6-tetrahydro-2,6-dioxo-7H-purinyl (e.g., 1,2,3,6-tetrahydro-1,3-dimethyl-2,6-dioxo-7H-purinyl); 1,2,3,6-tetrahydro-2,6-dioxo-1H-purinyl (e.g., 1,2,3,6-tetrahydro-3,7-dimethyl-2,6-dioxo-1H-purinyl); 2-oxobenz[c,d]indolyl; 1,1-dioxo-2H-naphth[1,8-c,d]isothiazolyl; and 1,8-naphthylenedicarboxamido. Additional heterocyclics include 3,3a,4,5,6,6a-hexahydro-pyrrolo[3,4-b]pyrrol-(2H)-yl, and 2,5-diazabicyclo[2.2.1]heptan-2-yl, homopiperazinyl (or diazepanyl), tetrahydropyranyl, dithiazolyl, benzofuranyl, benzothienyl, oxepanyl, thiepanyl, azocanyl, oxecanyl, and thiocanyl. Heterocyclic groups also include groups of the formula

where

E′ is selected from the group consisting of —N— and —CH—; F′ is selected from the group consisting of —N═CH—, —NH—CH₂—, —NH—C(O)—, —NH—, —CH═N—, —CH₂—NH—, —C(O)—NH—, —CH═CH—, —CH₂—, —CH₂CH₂—, —CH₂O—, —OCH₂—, —O—, and —S—; and G′ is selected from the group consisting of —CH— and —N—. Any of the heterocyclyl groups mentioned herein may be optionally substituted with one, two, three, four or five substituents independently selected from the group consisting of: (1) C₁₋₇ acyl (e.g., carboxyaldehyde); (2) C₁₋₂₀ alkyl (e.g., C₁₋₆ alkyl, C₁₋₆ alkoxy-C₁₋₆ alkyl, C₁₋₆ alkylsulfinyl-C₁₋₆ alkyl, amino-C₁₋₆ alkyl, azido-C₁₋₆ alkyl, (carboxyaldehyde)-C₁₋₆ alkyl, halo-C₁₋₆ alkyl (e.g., perfluoroalkyl), hydroxy-C₁₋₆ alkyl, nitro-C₁₋₆ alkyl, or C₁₋₆ thioalkoxy-C₁₋₆ alkyl); (3) C₁₋₂₀ alkoxy (e.g., C₁₋₆ alkoxy, such as perfluoroalkoxy); (4) C₁₋₆ alkylsulfinyl; (5) C₆₋₁₀ aryl; (6) amino; (7) C₁₋₆ alk-C₆₋₁₀ aryl; (8) azido; (9) C₃₋₈ cycloalkyl; (10) C₁₋₆ alk-C₃₋₈ cycloalkyl; (11) halo; (12) C₁₋₁₂ heterocyclyl (e.g., C₂-12 heteroaryl); (13) (C₁₋₁₂ heterocyclyl)oxy; (14) hydroxy; (15) nitro; (16) C₁₋₂₀ thioalkoxy (e.g., C₁₋₆ thioalkoxy); (17) —(CH₂)_(q)CO₂R^(A′), where q is an integer from zero to four, and R^(A′) is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, (c) hydrogen, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (18) —(CH₂)_(q)CONR^(B′)R^(C′), where q is an integer from zero to four and where R^(B′) and R^(C′) are independently selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (19) —(CH₂)_(q)SO₂R^(D′), where q is an integer from zero to four and where R^(D′) is selected from the group consisting of (a) C₁₋₆ alkyl, (b) C₆₋₁₀ aryl, and (c) C₁₋₆ alk-C₆₋₁₀ aryl; (20) —(CH₂)_(q)SO₂NR^(E′)R^(F′), where q is an integer from zero to four and where each of R^(E′) and R^(F′) is, independently, selected from the group consisting of (a) hydrogen, (b) C₁₋₆ alkyl, (c) C₆₋₁₀ aryl, and (d) C₁₋₆ alk-C₆₋₁₀ aryl; (21) thiol; (22) C₆₋₁₀ aryloxy; (23) C₃₋₈ cycloalkoxy; (24) arylalkoxy; (25) C₁₋₆ alk-C₁₋₁₂ heterocyclyl (e.g., C₁₋₆ alk-C₁₋₁₂ heteroaryl); (26) oxo; (27) (C₁₋₁₂ heterocyclyl)imino; (28) C₂₋₂₀ alkenyl; and (29) C₂₋₂₀ alkynyl. In some embodiments, each of these groups can be further substituted as described herein. For example, the alkylene group of a C₁-alkaryl or a C₁-alkheterocyclyl can be further substituted with an oxo group to afford the respective aryloyl and (heterocyclyl)oyl substituent group.

The term “hydrocarbon,” as used herein, represents a group consisting only of carbon and hydrogen atoms.

The term “hydroxyl,” as used herein, represents an —OH group. In some embodiments, the hydroxyl group can be substituted with 1, 2, 3, or 4 substituent groups (e.g., O-protecting groups) as defined herein for an alkyl.

The term “isomer,” as used herein, means any tautomer, stereoisomer, enantiomer, or diastereomer of any compound of the invention. It is recognized that the compounds of the invention can have one or more chiral centers and/or double bonds and, therefore, exist as stereoisomers, such as double-bond isomers (i.e., geometric E/Z isomers) or diastereomers (e.g., enantiomers (i.e., (+) or (−)) or cis/trans isomers). According to the invention, the chemical structures depicted herein, and therefore the compounds of the invention, encompass all of the corresponding stereoisomers, that is, both the stereomerically pure form (e.g., geometrically pure, enantiomerically pure, or diastereomerically pure) and enantiomeric and stereoisomeric mixtures, e.g., racemates. Enantiomeric and stereoisomeric mixtures of compounds of the invention can typically be resolved into their component enantiomers or stereoisomers by well-known methods, such as chiral-phase gas chromatography, chiral-phase high performance liquid chromatography, crystallizing the compound as a chiral salt complex, or crystallizing the compound in a chiral solvent. Enantiomers and stereoisomers can also be obtained from stereomerically or enantiomerically pure intermediates, reagents, and catalysts by well-known asymmetric synthetic methods.

The term “N-protected amino,” as used herein, refers to an amino group, as defined herein, to which is attached one or two N-protecting groups, as defined herein.

The term “N-protecting group,” as used herein, represents those groups intended to protect an amino group against undesirable reactions during synthetic procedures. Commonly used N-protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” 3^(rd) Edition (John Wiley & Sons, New York, 1999), which is incorporated herein by reference. N-protecting groups include acyl, aryloyl, or carbamyl groups such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl, and chiral auxiliaries such as protected or unprotected D, L or D, L-amino acids such as alanine, leucine, phenylalanine, and the like; sulfonyl-containing groups such as benzenesulfonyl, p-toluenesulfonyl, and the like; carbamate forming groups such as benzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl, p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl, 3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl, 4-methoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl, 3,4,5-trimethoxybenzyloxycarbonyl, 1-(p-biphenylyl)-1-methylethoxycarbonyl, α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxy carbonyl, t-butyloxycarbonyl, diisopropylmethoxycarbonyl, isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl, 2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxy carbonyl, fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl, adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl, and the like, alkaryl groups such as benzyl, triphenylmethyl, benzyloxymethyl, and the like and silyl groups, such as trimethylsilyl, and the like. Preferred N-protecting groups are formyl, acetyl, benzoyl, pivaloyl, t-butylacetyl, alanyl, phenylsulfonyl, benzyl, t-butyloxycarbonyl (Boc), and benzyloxycarbonyl (Cbz).

The term “O-protecting group,” as used herein, represents those groups intended to protect an oxygen containing (e.g., phenol, hydroxyl, or carbonyl) group against undesirable reactions during synthetic procedures. Commonly used O-protecting groups are disclosed in Greene, “Protective Groups in Organic Synthesis,” 3^(rd) Edition (John Wiley & Sons, New York, 1999), which is incorporated herein by reference. Exemplary O-protecting groups include acyl, aryloyl, or carbamyl groups, such as formyl, acetyl, propionyl, pivaloyl, t-butylacetyl, 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl, trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, α-chlorobutyryl, benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, t-butyldimethylsilyl, tri-iso-propylsilyloxymethyl, 4,4′-dimethoxytrityl, isobutyryl, phenoxyacetyl, 4-isopropylpehenoxyacetyl, dimethylformamidino, and 4-nitrobenzoyl; alkylcarbonyl groups, such as acyl, acetyl, propionyl, pivaloyl, and the like; optionally substituted arylcarbonyl groups, such as benzoyl; silyl groups, such as trimethylsilyl (TMS), tert-butyldimethylsilyl (TBDMS), tri-iso-propylsilyloxymethyl (TOM), triisopropylsilyl (TIPS), and the like; ether-forming groups with the hydroxyl, such methyl, methoxymethyl, tetrahydropyranyl, benzyl, p-methoxybenzyl, trityl, and the like; alkoxycarbonyls, such as methoxycarbonyl, ethoxycarbonyl, isopropoxycarbonyl, n-isopropoxycarbonyl, n-butyloxycarbonyl, isobutyloxycarbonyl, sec-butyloxycarbonyl, t-butyloxycarbonyl, 2-ethylhexyloxycarbonyl, cyclohexyloxycarbonyl, methyloxycarbonyl, and the like; alkoxyalkoxycarbonyl groups, such as methoxymethoxycarbonyl, ethoxymethoxycarbonyl, 2-methoxyethoxycarbonyl, 2-ethoxyethoxycarbonyl, 2-butoxyethoxycarbonyl, 2-methoxyethoxymethoxycarbonyl, allyloxycarbonyl, propargyloxycarbonyl, 2-butenoxycarbonyl, 3-methyl-2-butenoxycarbonyl, and the like; haloalkoxycarbonyls, such as 2-chloroethoxycarbonyl, 2-chloroethoxycarbonyl, 2,2,2-trichloroethoxycarbonyl, and the like; optionally substituted arylalkoxycarbonyl groups, such as benzyloxycarbonyl, p-methylbenzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, 2,4-dinitrobenzyloxycarbonyl, 3,5-dimethylbenzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-bromobenzyloxy-carbonyl, fluorenylmethyloxycarbonyl, and the like; and optionally substituted aryloxycarbonyl groups, such as phenoxycarbonyl, p-nitrophenoxycarbonyl, o-nitrophenoxycarbonyl, 2,4-dinitrophenoxycarbonyl, p-methylphenoxycarbonyl, m-methylphenoxycarbonyl, o-bromophenoxycarbonyl, 3,5-dimethylphenoxycarbonyl, p-chlorophenoxycarbonyl, 2-chloro-4-nitrophenoxy-carbonyl, and the like); substituted alkyl, aryl, and alkaryl ethers (e.g., trityl; methylthiomethyl; methoxymethyl; benzyloxymethyl; siloxymethyl; 2,2,2,-trichloroethoxymethyl; tetrahydropyranyl; tetrahydrofuranyl; ethoxyethyl; 1-[2-(trimethylsilyl)ethoxy]ethyl; 2-trimethylsilylethyl; t-butyl ether; p-chlorophenyl, p-methoxyphenyl, p-nitrophenyl, benzyl, p-methoxybenzyl, and nitrobenzyl); silyl ethers (e.g., trimethylsilyl; triethylsilyl; triisopropylsilyl; dimethylisopropylsilyl; t-butyldimethylsilyl; t-butyldiphenylsilyl; tribenzylsilyl; triphenylsilyl; and diphenymethylsilyl); carbonates (e.g., methyl, methoxymethyl, 9-fluorenylmethyl; ethyl; 2,2,2-trichloroethyl; 2-(trimethylsilyl)ethyl; vinyl, allyl, nitrophenyl; benzyl; methoxybenzyl; 3,4-dimethoxybenzyl; and nitrobenzyl); carbonyl-protecting groups (e.g., acetal and ketal groups, such as dimethyl acetal, 1,3-dioxolane, and the like; acylal groups; and dithiane groups, such as 1,3-dithianes, 1,3-dithiolane, and the like); carboxylic acid-protecting groups (e.g., ester groups, such as methyl ester, benzyl ester, t-butyl ester, orthoesters, and the like; and oxazoline groups.

The term “oxo” as used herein, represents ═O.

The term “polyethylene glycol,” as used herein, represents an alkoxy chain comprised of one or more monomer units, each monomer unit consisting of —OCH₂CH₂—. Polyethyelene glycol (PEG) is also sometimes referred to as polyethylene oxide (PEO) or polyoxyethylene (POE), and these terms may be considered interchangeable for the purpose of this invention. For example, a polyethylene glycol may have the structure, —(CH₂)_(s2)(OCH₂CH₂)_(s1)(CH₂)_(s3)O—, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), and each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10). Polyethylene glycol may also be considered to include an amino-polyethylene glycol of —NR^(N1)(CH₂)_(s2)(CH₂CH₂O)_(s1)(CH₂)_(s3)NR^(N1)—, wherein s1 is an integer from 1 to 10 (e.g., from 1 to 6 or from 1 to 4), each of s2 and s3, independently, is an integer from 0 to 10 (e.g., from 0 to 4, from 0 to 6, from 1 to 4, from 1 to 6, or from 1 to 10), and each R^(N1) is, independently, hydrogen or optionally substituted C₁₋₆ alkyl.

The term “stereoisomer,” as used herein, refers to all possible different isomeric as well as conformational forms which a compound may possess (e.g., a compound of any formula described herein), in particular all possible stereochemically and conformationally isomeric forms, all diastereomers, enantiomers and/or conformers of the basic molecular structure. Some compounds of the present invention may exist in different tautomeric forms, all of the latter being included within the scope of the present invention.

The term “sulfonyl,” as used herein, represents an —S(O)₂— group.

The term “thiol,” as used herein represents an —SH group.

Definitions

As used herein, the term “administered in combination” or “combined administration” means that two or more agents are administered to a subject at the same time or within an interval such that there may be an overlap of an effect of each agent on the patient. In some embodiments, they are administered within 90 days (e.g., within 80, 70, 60, 50, 40, 30, 20, 10, 5, 4, 3, 2, or 1 day(s)), within 28 days (e.g., with 14, 7, 6, 5, 4, 3, 2, or 1 day(s), within 24 hours (e.g., 12, 6, 5, 4, 3, 2, or 1 hour(s), or within about 60, 30, 15, 10, 5, or 1 minute of one another. In some embodiments, the administrations of the agents are spaced sufficiently closely together such that a combinatorial (e.g., a synergistic) effect is achieved.

As used herein, “antibody” refers to a polypeptide whose amino acid sequence including immunoglobulins and fragments thereof which specifically bind to a designated antigen, or fragments thereof. Antibodies in accordance with the present invention may be of any type (e.g., IgA, IgD, IgE, IgG, or IgM) or subtype (e.g., IgA1, IgA2, IgG1, IgG2, IgG3, or IgG4). Those of ordinary skill in the art will appreciate that a characteristic sequence or portion of an antibody may include amino acids found in one or more regions of an antibody (e.g., variable region, hypervariable region, constant region, heavy chain, light chain, and combinations thereof). Moreover, those of ordinary skill in the art will appreciate that a characteristic sequence or portion of an antibody may include one or more polypeptide chains, and may include sequence elements found in the same polypeptide chain or in different polypeptide chains.

As used herein, “antigen-binding fragment” refers to a portion of an antibody that retains the binding characteristics of the parent antibody.

The terms “bifunctional chelate” or “bifunctional conjugate” as used interchangeably herein, refer to a compound that contains a chelating group or metal complex thereof, a linker group, and a therapeutic moiety, targeting moiety, or cross linking group.

The term “cancer” refers to any cancer caused by the proliferation of malignant neoplastic cells, such as tumors, neoplasms, carcinomas, sarcomas, leukemias, and lymphomas. A “solid tumor cancer” is a cancer comprising an abnormal mass of tissue, e.g., sarcomas, carcinomas, and lymphomas. A “hematological cancer” or “liquid cancer,” as used interchangeably herein, is a cancer present in a body fluid, e.g., lymphomas and leukemias.

The term “chelate” as used herein, refers to an organic compound or portion thereof that can be bonded to a central metal or radiometal atom at two or more points.

The term “conjugate,” as used herein, refers to a molecule that contains a chelating group or metal complex thereof, a linker group, and which optionally contains a therapeutic moiety, targeting moiety, or cross linking group.

As used herein, the term “compound,” is meant to include all stereoisomers, geometric isomers, and tautomers of the structures depicted.

The compounds described herein can be asymmetric (e.g., having one or more stereocenters). All stereoisomers, such as enantiomers and diastereomers, are intended unless otherwise indicated. Compounds of the present disclosure that contain asymmetrically substituted carbon atoms can be isolated in optically active or racemic forms. Methods on how to prepare optically active forms from optically active starting materials are known in the art, such as by resolution of racemic mixtures or by stereoselective synthesis. Many geometric isomers of olefins, C═N double bonds, and the like can also be present in the compounds described herein, and all such stable isomers are contemplated in the present disclosure. Cis and trans geometric isomers of the compounds of the present disclosure are described and may be isolated as a mixture of isomers or as separated isomeric forms.

Compounds of the present disclosure also include tautomeric forms. Tautomeric forms result from the swapping of a single bond with an adjacent double bond and the concomitant migration of a proton. Tautomeric forms include prototropic tautomers which are isomeric protonation states having the same empirical formula and total charge. Examples prototropic tautomers include ketone—enol pairs, amide—imidic acid pairs, lactam—lactim pairs, amide—imidic acid pairs, enamine—imine pairs, and annular forms where a proton can occupy two or more positions of a heterocyclic system, such as, 1H- and 3H-imidazole, 1H-, 2H- and 4H-1,2,4-triazole, 1H- and 2H-isoindole, and 1H- and 2H-pyrazole. Tautomeric forms can be in equilibrium or sterically locked into one form by appropriate substitution.

At various places in the present specification, substituents of compounds of the present disclosure are disclosed in groups or in ranges. It is specifically intended that the present disclosure include each and every individual subcombination of the members of such groups and ranges. For example, the term “C₁₋₆ alkyl” is specifically intended to individually disclose methyl, ethyl, C₃ alkyl, C₄ alkyl, C₅ alkyl, and C₆ alkyl. Herein a phrase of the form “optionally substituted X” (e.g., optionally substituted alkyl) is intended to be equivalent to “X, wherein X is optionally substituted” (e.g., “alkyl, wherein said alkyl is optionally substituted”). It is not intended to mean that the feature “X” (e.g. alkyl) per se is optional.

As used herein “detection agent” refers to a molecule or atom which is useful in diagnosing a disease by locating the cells containing the antigen. Various methods of labeling polypeptides with detection agents are known in the art. Examples of detection agents include, but are not limited to, radioisotopes and radionuclides, dyes (such as with the biotin-streptavidin complex), contrast agents, luminescent agents (e.g., FITC, rhodamine, lanthanide phosphors, cyanine, and near IR dyes), and magnetic agents, such as gadolinium chelates.

As used herein, the term “radionuclide,” refers to an atom capable of undergoing radioactive decay (e.g., ³H, ¹⁴C, ¹⁵N, ¹⁸F, ³⁵S, ⁴⁷Sc, ⁵⁵Co, ⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁸⁹Zr, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y, ⁹⁷Ru, ⁹⁹Tc, ^(99m)Tc ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁴⁹Pm, ¹⁴⁹Tb, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁸Au, ¹⁹⁹Au, ²⁰³Pb, ²¹¹At, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²³Ra, ²²⁵Ac, ²²⁷Th, ²²⁹Th ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁸²Rb, ^(117m)Sn, ²⁰¹Tl). The terms radioactive nuclide, radioisotope, or radioactive isotope may also be used to describe a radionuclide. Radionuclides may be used as detection agents, as described above. In some embodiments, the radionuclide may be an alpha-emitting radionuclide.

The term an “effective amount” of an agent (e.g., any of the foregoing conjugates), as used herein, is that amount sufficient to effect beneficial or desired results, such as clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied.

The term “immunoconjugate,” as used herein, refers to a conjugate that includes a targeting moiety, such as an antibody, nanobody, affibody, or a consensus sequence from Fibronectin type III domain. In some embodiments, the immunoconjugate comprises an average of at least 0.10 conjugates per targeting moiety (e.g., an average of at least 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 4, 5, or 8 conjugates per targeting moiety).

The term “radioconjugate,” as used herein, refers to any conjugate that includes a radioisotope or radionuclide, such as any of the radioisotopes or radionuclides described herein.

The term “radioimmunoconjugate,” as used herein, refers to any immunoconjugate that includes a radioisotope or radionuclide, such as any of the radioisotopes or radionuclides described herein.

The term “radioimmunotherapy,” as used herein, refers a method of using a radioimmunoconjugate to produce a therapeutic effect. In some embodiments, radioimmunotherapy may include administration of a radioimmunoconjugate to a subject in need thereof, wherein administration of the radioimmunoconjugate produces a therapeutic effect in the subject. In some embodiments, radioimmunotherapy may include administration of a radioimmunoconjugate to a cell, wherein administration of the radioimmunoconjugate kills the cell. Wherein radioimmunotherapy involves the selective killing of a cell, in some embodiments the cell is a cancer cell in a subject having cancer.

The term “pharmaceutical composition,” as used herein, represents a composition containing a compound described herein formulated with a pharmaceutically acceptable excipient. In some embodiments, the pharmaceutical composition is manufactured or sold with the approval of a governmental regulatory agency as part of a therapeutic regimen for the treatment of disease in a mammal. Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., a tablet, capsule, caplet, gelcap, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a sterile solution free of particulate emboli and in a solvent system suitable for intravenous use); or in any other formulation described herein.

A “pharmaceutically acceptable excipient,” as used herein, refers any ingredient other than the compounds described herein (for example, a vehicle capable of suspending or dissolving the active compound) and having the properties of being nontoxic and non-inflammatory in a patient. Excipients may include, for example: antiadherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colors), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (flow enhancers), lubricants, preservatives, printing inks, radioprotectants, sorbents, suspending or dispersing agents, sweeteners, or waters of hydration. Exemplary excipients include, but are not limited to: ascorbic acid, histidine, phosphate buffer, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, crosslinked polyvinyl pyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropyl cellulose, hydroxypropyl methylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methyl paraben, microcrystalline cellulose, polyethylene glycol, polyvinyl pyrrolidone, povidone, pregelatinized starch, propyl paraben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethyl cellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

The term “pharmaceutically acceptable salt,” as use herein, represents those salts of the compounds described here that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without undue toxicity, irritation, or allergic response. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in: Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and in Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P. H. Stahl and C. G. Wermuth), Wiley-VCH, 2008. The salts can be prepared in situ during the final isolation and purification of the compounds described herein or separately by reacting the free base group with a suitable organic acid.

The compounds of the invention may have ionizable groups so as to be capable of preparation as pharmaceutically acceptable salts. These salts may be acid addition salts involving inorganic or organic acids or the salts may, in the case of acidic forms of the compounds of the invention be prepared from inorganic or organic bases. Frequently, the compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases are well-known in the art, such as hydrochloric, sulphuric, hydrobromic, acetic, lactic, citric, or tartaric acids for forming acid addition salts, and potassium hydroxide, sodium hydroxide, ammonium hydroxide, caffeine, various amines for forming basic salts. Methods for preparation of the appropriate salts are well-established in the art.

Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, toluenesulfonate, undecanoate, valerate salts, among others. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as nontoxic ammonium, quaternary ammonium, and amine cations, including, but not limited to ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

The term “therapeutic moiety” as used herein refers to any molecule or any part of a molecule that confers a therapeutic benefit. In some embodiments, the therapeutic moiety is a protein or polypeptide, e.g., an antibody, an antigen-binding fragment thereof. In some embodiments, the therapeutic moiety is a small molecule.

The term “targeting moiety” as used herein refers to any molecule or any part of a molecule that binds to a given target. In some embodiments, the targeting moiety is a protein or polypeptide such as an antibody or antigen binding fragment thereof, a nanobody, an affibody, or a consensus sequence from a Fibronectin type III domain.

The term “cross-linking group” as used herein refers to any reactive group that is able to join two or more molecules by a covalent bond. In some embodiments, the cross-linking group is an amino-reactive or thiol-reactive cross-linking group. In some embodiments, the amino-reactive or thiol-reactive cross-linking group comprises an activated ester such as a hydroxysuccinimide ester, 2,3,5,6-tetrafluorophenol ester, 4-nitrophenol ester or an imidate, anhydride, thiol, disulfide, maleimide, azide, alkyne, strained alkyne, strained alkene, halogen, sulfonate, haloacetyl, amine, hydrazide, diazirine, phosphine, tetrazine, isothiocyanate. In some embodiments, the cross linking group may be glycine-glycine-glycine and/or leucine-proline-(any amino acid)-threonine-glycine (SEQ ID NO: 11), which are the recognition sequences for coupling targeting agents with the linker using a sortase-mediated coupling reaction. The person having ordinary skill in the art will understand that the use of cross linking groups in the practice of the invention are not limited to the specific constructs disclosed herein, but rather may include other known cross linking groups.

The term “polypeptide” as used herein refers to a string of at least two amino acids attached to one another by a peptide bond. In some embodiments, a polypeptide may include at least 3-5 amino acids, each of which is attached to others by way of at least one peptide bond. Those of ordinary skill in the art will appreciate that polypeptides can include one or more “non-natural” amino acids or other entities that nonetheless are capable of integrating into a polypeptide chain. In some embodiments, a polypeptide may be glycosylated, e.g., a polypeptide may contain one or more covalently linked sugar moieties. In some embodiments, a single “polypeptide” (e.g., an antibody polypeptide) may comprise two or more individual polypeptide chains, which may in some cases be linked to one another, for example by one or more disulfide bonds or other means.

By “subject” is meant a human or non-human animal (e.g., a mammal).

By “substantial identity” or “substantially identical” is meant a polypeptide sequence that has the same polypeptide sequence, respectively, as a reference sequence, or has a specified percentage of amino acid residues, respectively, that are the same at the corresponding location within a reference sequence when the two sequences are optimally aligned. For example, an amino acid sequence that is “substantially identical” to a reference sequence has at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the reference amino acid sequence. For polypeptides, the length of comparison sequences will generally be at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 75, 90, 100, 150, 200, 250, 300, or 350 contiguous amino acids (e.g., a full-length sequence). Sequence identity may be measured using sequence analysis software on the default setting (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705). Such software may match similar sequences by assigning degrees of homology to various substitutions, deletions, and other modifications.

As used herein, and as well understood in the art, “to treat” a condition or “treatment” of the condition (e.g., the conditions described herein such as cancer) is an approach for obtaining beneficial or desired results, such as clinical results. Beneficial or desired results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions; diminishment of extent of disease, disorder, or condition; stabilized (i.e., not worsening) state of disease, disorder, or condition; preventing spread of disease, disorder, or condition; delay or slowing the progress of the disease, disorder, or condition; amelioration or palliation of the disease, disorder, or condition; and remission (whether partial or total), whether detectable or undetectable. “Palliating” a disease, disorder, or condition means that the extent and/or undesirable clinical manifestations of the disease, disorder, or condition are lessened and/or time course of the progression is slowed or lengthened, as compared to the extent or time course in the absence of treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depicting the general structure of a conjugate comprising a chelate, a linker, and a cross-linking group (top) and a conjugate comprising a chelate, a linker, and a targeting moiety (bottom).

FIG. 2 is a schematic depicting the synthesis of the bifunctional chelate, 4-{[11-oxo-11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound B). The synthesis of Compound B is described in Example 3.

FIG. 3 is a schematic depicting the synthesis of the bifunctional chelate, 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound C). The synthesis of Compound C is described in Example 4.

FIG. 4 is a graph depicting the percent residualization of three bifunctional chelated antibodies (Compound A, Compound B, and Compound C) determined as CPM (lysate)/CPM (efflux+recycled+lysate). The residualization assay used is described in detail in Example 6.

FIG. 5 is a series of graphs depicting the metabolic excretion profile of non-targeted human IgG antibody conjugates [¹⁷⁷Lu]-Compound B-HuMIGF-1R, and [¹⁷⁷Lu]-Compound C-HuMIGF-1R as compared to [¹⁷⁷Lu]-Compound A-HuMIGF-1R, the methods and results of which are described in detail in Example 9.

FIG. 6. Blood Pharmacokinetics of Total Radioactivity in CD-1 Nude Mice. Results and methods are described in Example 9.

FIG. 7. Therapeutic efficacy of [²²⁵Ac]-HuMIGF-1R Compounds (200 nCi dose). Results and methods are described in Example 10.

FIG. 8 is a series of graphs depicting the metabolic excretion profile of non-targeted human IgG antibody conjugates [¹⁷⁷Lu]-Compound B-HuMIgG, and [¹⁷⁷Lu]-Compound C-HuMIgG as compared to [¹⁷⁷Lu]-Compound A-HuMIgG, the methods and results of which are described in detail in Example 14.

FIG. 9 is a graph showing the biodistribution of radiolabeled [²²⁵Ac]-Compound C-HuMIGF-1R. Results and methods are described in Example 24.

FIG. 10 is a graph showing the biodistribution of radiolabeled [¹⁷⁷Lu]-Compound C-HuMIGF-1R. Results and methods are described in Example 24.

FIG. 11 is a graph showing the biodistribution of radiolabeled [¹¹¹In]-Compound C-HuMIGF-1R. Results and methods are described in Example 24.

FIG. 12 is a graph showing the tumor uptake of [¹⁷⁷Lu]-Compound C-HuMIGF-1R in Colo-205 tumor xenograft bearing mice. Mice (n=4/group) received [¹⁷⁷Lu]-Compound C-HuMIGF-1R with increasing amounts of antibody HuMIGF-1R administered simultaneously (“Block”). Results and methods are described in Example 25.

FIG. 13 is a graph showing the therapeutic efficacy of [²²⁵Ac]-Compound C-HuMIGF-1R in mice bearing colorectal (Colo-205) tumor xenografts (Initial Tumor Volume ˜200 mm³). Statistics by ANOVA with Dunnett's Multiple Comparison against vehicle control. Results and methods are described in Example 26.

FIG. 14 is a graph showing the therapeutic efficacy of [²²⁵Ac]-Compound C-HuMIGF-1R in mice bearing prostate (LNCaP) tumor xenografts (Initial Tumor Volume ˜200 mm³). Statistics by ANOVA with Dunnett's Multiple Comparison against vehicle control. Results and methods are described in Example 26.

FIG. 15 is a graph showing the therapeutic efficacy of [²²⁵Ac]-Compound C-HuMIGF-1R in mice bearing radioresistant lung (A549) tumor xenografts (Initial Tumor Volume ˜200 mm³). Statistics by ANOVA with Dunnett's Multiple Comparison against vehicle control. Results and methods are described in Example 26.

FIG. 16 is a graph showing the therapeutic efficacy of [²²⁵Ac]-Compound C-HuMIGF-1R in mice bearing large colorectal (Colo-205) tumor xenografts (Initial Tumor Volume >400 mm³). Statistics by ANOVA with Dunnett's Multiple Comparison against vehicle control. Results and methods are described in Example 26.

FIG. 17 is a graph showing the therapeutic efficacy of [²²⁵Ac]-Compound C-HuMIGF-1R compared to unconjugated antibody HuMIGF-1R in mice bearing colorectal (Colo-205) tumor xenografts. Statistics by ANOVA with Dunnett's Multiple Comparison against vehicle control. Results and methods are described in Example 26.

FIG. 18 is a graph showing the therapeutic efficacy of [²²⁵Ac]-Compound C-HuMIGF-1R compared to unconjugated antibody HuMIGF-1R in mice bearing radioresistant lung (A549) tumor xenografts. Statistics by ANOVA with Dunnett's Multiple Comparison against vehicle control. Results and methods are described in Example 26.

FIG. 19 is a graph showing the extended therapeutic efficacy, measured as relative tumor volume, of [²²⁵Ac]-Compound C-HuMIGF-1R in mice bearing colorectal (Colo-205) tumor xenografts.

Statistics by ANOVA with Dunnett's Multiple Comparison against vehicle control. Results and methods are described in Example 27.

FIG. 20 is a graph showing the extended therapeutic efficacy, measured as percent survival, of [²²⁵Ac]-Compound C-HuMIGF-1R in mice bearing colorectal (Colo-205) tumor xenografts. Statistics by ANOVA with Dunnett's Multiple Comparison against vehicle control. Results and methods are described in Example 27.

FIG. 21 is a graph showing the efficacy of dose fractioned [²²⁵Ac]-Compound C-HuMIGF-1R in mice bearing colorectal cancer (Colo-205) tumor xenografts. Statistics by ANOVA with Dunnett's Multiple Comparison against vehicle control. Results and methods are described in Example 28.

FIG. 22 is a graph showing the efficacy of dose fractioned [²²⁵Ac]-Compound C-HuMIGF-1R in mice bearing radioresistant lung (A549) tumor xenografts. Statistics by ANOVA with Dunnett's Multiple Comparison against vehicle control. Results and methods are described in Example 28.

DETAILED DESCRIPTION

Radiolabelled targeting moieties (also known as radioimmunoconjugates) are designed to target a protein or receptor that is upregulated in a disease state to deliver a radioactive payload to damage and kill cells of interest (radioimmunotherapy). The process of delivering such a payload, via radioactive decay, produces an alpha, beta, or gamma particle or Auger electron that can cause direct effects to DNA (such as single or double stranded DNA breaks) or indirect effects such as by-stander or crossfire effects.

Radioimmunoconjugates typically contain a biological targeting moiety (e.g, an antibody or antigen binding fragment thereof that specifically binds to IGF-1R), a radioisotope, and a molecule that links the two. Conjugates are formed when a bifunctional chelate is appended to the biological targeting molecule so that structural alterations are minimal while maintaining target affinity. Once radiolabelled, the final radioimmunoconjugate is formed.

Bifunctional chelates structurally contain a chelate, the linker, and cross-linking group (FIG. 1). When developing new bifunctional chelates, most efforts focus around the chelating portion of the molecule. Several examples of bifunctional chelates have been described with various cyclic and acyclic structures conjugated to a targeted moiety. [Bioconjugate Chem. 2000, 11, 510-519, Bioconjugate Chem. 2012, 23, 1029-1039, Mol Imaging Biol (2011) 13:215-221, Bioconjugate Chem. 2002, 13, 110-115]

One of the key factors of developing safe and effective radioimmunoconjugates is maximizing efficacy while minimizing off-target toxicity in normal tissue. While this statement is one of the core tenants of developing new drugs, the application to radioimmunotherapeutics presents new challenges. Radioimmunoconjugates do not need to block a receptor, as needed with a therapeutic antibody, or release the cytotoxic payload intracellularly, as required with an antibody drug conjugate, in order to have therapeutic efficacy. However, the emission of the toxic particle is an event that occurs as a result of first-order (radioactive) decay and can occur at random anywhere inside the body after administration. Once the emission occurs, damage could occur to surrounding cells within the range of the emission leading to the potential of off-target toxicity. Therefore, limiting exposure of these emissions to normal tissue is the key to developing new drugs.

One potential method for reducing off-target exposure is to remove the radioactivity more effectively from the body (e.g., from normal tissue in the body). The most obvious mechanism is to increase the rate of clearance of the biological targeting agent. This approach likely requires identifying ways to shorten the half-life of the biological targeting agent, which is a topic not well described for biological targeting agents. Regardless of the mechanism, increasing drug clearance will also negatively impact the pharmacodynamics/efficacy in that the more rapid removal of drug from the body will lower the effective concentration at the site of action, which, in turn, would require a higher total dose and would not achieve the desired results of reducing total radioactive dose to normal tissue.

Other efforts have focused on accelerating the metabolism of the portion of the molecule that contains the radioactive moiety. To this end, some efforts have been made to increase the rate of cleavage of the radioactivity from the biological targeting agents using what have been termed “cleavable linkers”. Cleavable linkers, however, have been taken on different meaning as it relates to radioimmunoconjugates. Cornelissen, et al. has described cleavable linkers as those by which the bifunctional conjugate attaches to the biologic targeting agent through a reduced cysteine, whereas others have described the use of enzyme-cleavable systems that require the co-administration of the radioimmunoconjugate with a cleaving agent/enzyme to release [Mol Cancer Ther; 12(11) November 2013, Methods in Molecular Biology, 2009, 539, 191-211, Bioconjugate chemistry, Volume 14, Issue 5, p. 927-33 (2003)]. These methods either change the nature of the biological targeting moiety, in the case of the cysteine linkage, or are not practical from a drug development perspective (enzyme cleavable systems) since, in the case of the citations provided, require the administration of 2 agents.

The focus of the embodiments described herein centers on more effectively eliminating radioactivity from the body after catabolism and/or metabolism of the radioimmunoconjugate by making modifications to the linker region of the bifunctional chelate.

This is a novel approach since little information appears to exist describing the in vivo impact of the linker, especially as it applies to radioimmunoconjugates. One potential reason is that following catabolism/metabolism of the radioimmunoconjugate, one would expect the radiolabelled conjugate to undergo rapid systemic elimination. The supposition was furthered experimentally when the bifunctional chelate was administered alone; it cleared the bloodstream faster than the radioimmunoconjugate with that same bifunctional chelate. Based on this data, one would expect that following catabolism/metabolism of the radioimmunoconjugate, the metabolite containing the bifunctional chelate would also be rapidly eliminated.

However, rapid clearance of the metabolites containing the radiolabelled conjugate does not necessarily occur in vivo. Based on the results described below, the linker region of bifunctional chelates can directly impact the elimination of the radioactivity from the body following catabolism of the radioconjugate while not having a detrimental impact to the overall in vitro properties or the in vivo pharmacokinetics and pharmacodynamics of the radioimmunoconjugate. Data is presented below that demonstrates that certain bifunctional chelates available commercially produce a slower rate and a lower extent of elimination of the total radioactivity from the body when compared to the embodiments described herein.

Therefore, through the embodiments described herein, bifunctional chelates, when attached to biological targeting moieties or therapeutic agents, have been identified that achieve a reduction of total body radioactivity by increasing the extent of excretion of the catabolic/metabolic products while maintaining the pharmacokinetics of the intact molecule when compared to similar bifunctional chelates in the public domain. This reduction in total body radioactivity has been determined to be due to the clearance of catabolic/metabolic by-products and does not impact the other in vitro and in vivo properties such as degree of specificity (in vitro binding), cellular retention, and tumor uptake in vivo. When taken in whole, these embodiments achieve the desired properties of radioimmunoconjugates by reducing the body burden of radioactivity while maintaining on-target activity.

Therapeutic Moieties and Targeting Moieties

Therapeutic or targeting moieties include any molecule or any part of a molecule that confers a therapeutic benefit. In some embodiments, the therapeutic moiety is a protein or polypeptide, e.g., an antibody, an antigen-binding fragment thereof. In some embodiments, the therapeutic moiety is a small molecule. Targeting moieties include any molecule or any part of a molecule that binds to a given target. In some embodiments, the targeting moiety is a protein or polypeptide such as antibodies or antigen binding fragments thereof, nanobodies, affibodies, and consensus sequences from Fibronectin type III domains (e.g., Centyrins or Adnectins).

Polypeptides

Polypeptides include, for example, any of a variety of hematologic agents (including, for instance, erythropoietin, blood-clotting factors, etc.), interferons, colony stimulating factors, antibodies, enzymes, and hormones. The identity of a particular polypeptide is not intended to limit the present disclosure, and any polypeptide of interest can be a polypeptide in the present methods.

A reference polypeptide described herein can include a target-binding domain that binds to a target of interest (e.g., binds to an antigen). For example, a polypeptide, such as an antibody, can bind to a transmembrane polypeptide (e.g., receptor) or ligand (e.g., a growth factor). Exemplary molecular targets (e.g., antigens) for polypeptides described herein (e.g., antibodies) include CD proteins such as CD2, CD3, CD4, CD8, CD11, CD19, CD20, CD22, CD25, CD33, CD34, CD40, CD52; members of the ErbB receptor family such as the EGF receptor (EGFR, HER1, ErbB1), HER2 (ErbB2), HER3 (ErbB3) or HER4 (ErbB4) receptor; macrophage receptors such as CRIg; tumor necrosis factors such as TNFα or TRAIL/Apo-2; cell adhesion molecules such as LFA-1, Mac1, p150, 95, VLA-4, ICAM-1, VCAM and αvβ3 integrin including either α or β subunits thereof (e.g., anti-CD11a, anti-CD18 or anti-CD11 b antibodies); growth factors and receptors such as EGF, FGFR (e.g., FGFR3) and VEGF; IgE; cytokines such as IL1; cytokine receptors such as IL2 receptor; blood group antigens; flk2/flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; protein C; neutropilins; ephrins and receptors; netrins and receptors; slit and receptors; chemokines and chemokine receptors such as CCL5, CCR4, CCR5; amyloid beta; complement factors, such as complement factor D; lipoproteins, such as oxidized LDL (oxLDL); lymphotoxins, such as lymphotoxin alpha (LTa). Other molecular targets include Tweak, B7RP-1, proprotein convertase subtilisin/kexin type 9 (PCSK9), sclerostin, c-kit, Tie-2, c-fms, and anti-M1.

Antibodies

An IgG antibody consists of two identical light polypeptide chains and two identical heavy polypeptide chains linked together by disulfide bonds. The first domain located at the amino terminus of each chain is variable in amino acid sequence, providing the antibody binding specificities found in each individual antibody. These are known as variable heavy (VH) and variable light (VL) regions. The other domains of each chain are relatively invariant in amino acid sequence and are known as constant heavy (CH) and constant light (CL) regions. For an IgG antibody, the light chain includes one variable region (VL) and one constant region (CL). An IgG heavy chain includes a variable region (VH), a first constant region (CH1), a hinge region, a second constant region (CH2), and a third constant region (CH3). In IgE and IgM antibodies, the heavy chain includes an additional constant region (CH4).

Antibodies described herein can include, for example, monoclonal antibodies, polyclonal antibodies, multispecific antibodies, human antibodies, humanized antibodies, camelid antibodies, chimeric antibodies, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and antigen-binding fragments of any of the above. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass.

The term “antigen binding fragment” of an antibody, as used herein, refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Examples of binding fragments encompassed within the term “antigen binding fragment” of an antibody include a Fab fragment, a F(ab′)₂ fragment, a Fd fragment, a Fv fragment, a scFv fragment, a dAb fragment (Ward et al., (1989) Nature 341:544-546), and an isolated complementarity determining region (CDR). These antibody fragments can be obtained using conventional techniques known to those with skill in the art, and the fragments can be screened for utility in the same manner as are intact antibodies.

Antibodies or fragments described herein can be produced by any method known in the art for the synthesis of antibodies (see, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Brinkman et al., 1995, J. Immunol. Methods 182:41-50; WO 92/22324; WO 98/46645). Chimeric antibodies can be produced using the methods described in, e.g., Morrison, 1985, Science 229:1202, and humanized antibodies by methods described in, e.g., U.S. Pat. No. 6,180,370.

Additional antibodies described herein are bispecific antibodies and multivalent antibodies, as described in, e.g., Segal et al., J. Immunol. Methods 248:1-6 (2001); and Tutt et al., J. Immunol. 147: 60 (1991).

Insulin-Like Growth Factor 1 (IGF-1R) Antibodies

Insulin-like growth factor 1 receptor is a transmembrane protein found on the surface of human cells activated by insulin-like growth factor 1 (IGF-1) and 2 (IGF-2). Radioimmunoconjugates of the invention may include the insulin-like growth factor-1 receptor (IGF-1R). Although not a typical oncogene, IGF-1R promotes initiation and progression of cancer, playing a critical role in mitogenic transformation and maintenance of the transformed phenotype. IGF-1R has been associated with development of multiple common cancers including breast, lung (e.g., non-small lung), liver, prostate, pancreas, ovarian, colon, melanoma, adrenocortical carcinoma, and various types of sarcomas. IGF-1R signaling stimulates tumour cell proliferation and metabolism, supports angiogenesis, and confers protection from apoptosis. It affects metastatic factors (e.g. HIF-1 dependent hypoxia signaling), anchorage independent growth, as well as growth and survival of tumour metastases after extravasation. IGF-1R has also been implicated in the development, maintenance and enrichment of therapeutic resistant cancer stem cell populations.

Despite the abundance of data implicating IGF-1R's role in cancer, therapeutics targeting IGF-1R have yet to demonstrate a significant impact on disease. There has been much speculation for this lack of efficacy including the inability to identify appropriate biomarkers for patient identification, complexity and interdependency of the IGF-1/IR signaling pathway and the development of other growth hormone compensatory mechanisms [Beckwith and Yee, Mol Endocrinol, November 2015, 29(11):1549-1557]. Radioimmunotherapy, however, may provide a viable mechanism for treating cancers over expressing the IGF-1 receptor by utilizing the ability of IGF-1R to undergo antibody triggered internalization and lysosomal degradation to deliver targeted radioisotopes inside cancer cells. Internalization and lysosomal degradation of an IGF-1R targeted radioimmunoconjugate prolongs the residence time of the delivered radioisotope inside cancer cells thereby maximizing the potential for a cell killing emission to occur. In the case of actinium-225 which yields 4 alpha particles per decay chain, cell death can be accomplished by as little as 1 atom of radionuclide delivered per cell [Sgouros, et al. J Nucl Med. 2010, 51:311-2]. Cell killing due to direct DNA impact and breakage by an alpha particle may occur in the targeted cell or in a radius of 2 or 3 non-targeted cells for a given alpha particle decay. In addition to having very high potential anti-tumour potency, IGF-1R targeted radioimmunoconjugates may not generate mechanistic resistance as they do not rely on blocking ligand binding to the receptor to inhibit the oncologic process, as needed with a therapeutic antibody.

Several IGF-1R antibodies have been developed and investigated for the treatment of various types of cancers including figitumumab, cixutumumab, ganitumab, AVE1642 (also known as humanized EM164 and huEM164), BIIB002, robatumumab, and teprotumumab. After binding to IGF-1R, these antibodies are internalized into the cell and degraded by lysosomal enzymes. The combination of overexpression on tumor cells and internalization offers the possibility of delivering detection agents directly to the tumor site while limiting the exposure of normal tissues to toxic agents.

The CDRs of the light chain variable region of AVE1642 have the sequences:

SEQ ID NO: 1 (CDR-L1) RSSQSIVHSNVNTYLE

SEQ ID NO: 2 (CDR-L2) KVSNRFS

SEQ ID NO: 3 (CDR-L3) FQGSHVPPT

The light chain variable region of AVE1642 has the sequence:

SEQ ID NO: 4 DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPRL LIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAK

The CDRs of the heavy chain variable region of AVE1642 have the sequences:

SEQ ID NO: 5 (CDR-H1) SYWMH

SEQ ID NO: 6 (CDR-H2) GEINPSNGRTNY NQKFQG

SEQ ID NO: 7 (CDR-H3) GRPDYYGSSKWY FDV

The heavy chain variable region of AVE1642 has the sequence:

SEQ ID NO: 8 QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMHWVKQRPGQGLEWIGEI NPSNGRTNYNQKFQGKATLTVDKSSSTAYMQLSSLTSEDSAVYYFARGRPD YYGSSKWYFDVWGQGTTVTVSSASTKGPSVFPLAPSSKSTSGGTAALG

AVE1642 has the sequence:

SEQ ID NO: 9 DVVMTQTPLSLPVSLGDPASISCRSSQSIVHSNVNTYLEWYLQKPGQSPRL LIYKVSNRFSGVPDRFSGSGAGTDFTLRISRVEAEDLGIYYCFQGSHVPPT FGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQW KVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQ GLSSPVTKSFNRGECQVQLVQSGAEVVKPGASVKLSCKASGYTFTSYVVMH WVKQRPGQGLEWIGEINPSNGRTNYNQKFQGKATLTVDKSSSTAYMQLSSL TSEDSAVYYFARGRPDYYGSSKWYFDVWGQGTTVTVSSASTKGPSVFPLAP SSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS LSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVE VHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNG QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHY TQKSLSLSPGK

Nanobodies Nanobodies are antibody fragments consisting of a single monomeric variable antibody domain.

Nanobodies may also be referred to as single-domain antibodies. Like antibodies, nanobodies bind selectively to a specific antigen. Nanobodies may be heavy-chain variable domains or light chain domains. Nanobodies may occur naturally or be the product of biological engineering. Nanobodies may be biologically engineered by site-directed mutagenesis or mutagenic screening (e.g., phage display, yeast display, bacterial display, mRNA display, ribosome display).

Affibodies

Affibodies are polypeptides or proteins engineered to bind to a specific antigen. As such, affibodies may be considered to mimic certain functions of antibodies. Affibodies may be engineered variants of the B-domain in the immunoglobulin-binding region of staphylococcal protein A. Affibodies may be engineered variants of the Z-domain, a B-domain that has lower affinity for the Fab region. Affibodies may be biologically engineered by site-directed mutagenesis or mutagenic screening (e.g., phage display, yeast display, bacterial display, mRNA display, ribosome display).

Affibody molecules showing specific binding to a variety of different proteins (e.g. insulin, fibrinogen, transferrin, tumor necrosis factor-α, IL-8, gp120, CD28, human serum albumin, IgA, IgE, IgM, HER2 and EGFR) have been generated, demonstrating affinities (K_(d)) in the μM to pM range.

Fibronectin Type III Domains

The Fibronectin type III domain is an evolutionarily conserved protein domain found in a wide-variety of extracellular proteins. The Fibronectin type III domain has been used as a molecular scaffold to produce molecules capable of selectively binding a specific antigen. Variants of the Fibronectin type III domains (FN3) that have been engineered for selective-binding may also be referred to as monobodies. FN3 domains may be biologically engineered by site-directed mutagenesis or mutagenic screening (e.g., CIS-display, phage display, yeast display, bacterial display, mRNA display, ribosome display).

Modified Polypeptides

The polypeptides of the invention may have a modified amino acid sequence. Modified polypeptides may be substantially identical to the corresponding reference polypeptide (e.g., the amino acid sequence of the modified polypeptide may have at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of the reference polypeptide). In certain embodiments, the modification does not destroy significantly a desired biological activity (e.g., binding to IGF-1R). The modification may reduce (e.g., by at least 5%, 10%, 20%, 25%, 35%, 50%, 60%, 70%, 75%, 80%, 90%, or 95%), may have no effect, or may increase (e.g., by at least 5%, 10%, 25%, 50%, 100%, 200%, 500%, or 1000%) the biological activity of the original polypeptide. The modified polypeptide may have or may optimize a characteristic of a polypeptide, such as in vivo stability, bioavailability, toxicity, immunological activity, immunological identity, and conjugation properties.

Modifications include those by natural processes, such as post-translational processing, or by chemical modification techniques known in the art. Modifications may occur anywhere in a polypeptide including the polypeptide backbone, the amino acid side chains and the amino- or carboxy-terminus. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide, and a polypeptide may contain more than one type of modification. Polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from post-translational natural processes or may be made synthetically. Other modifications include pegylation, acetylation, acylation, addition of acetomidomethyl (Acm) group, ADP-ribosylation, alkylation, amidation, biotinylation, carbamoylation, carboxyethylation, esterification, covalent attachment to flavin, covalent attachment to a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of drug, covalent attachment of a marker (e.g., fluorescent or radioactive), covalent attachment of a lipid or lipid derivative, covalent attachment of phosphatidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cystine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation and ubiquitination.

A modified polypeptide can also include an amino acid insertion, deletion, or substitution, either conservative or non-conservative (e.g., D-amino acids, desamino acids) in the polypeptide sequence (e.g., where such changes do not substantially alter the biological activity of the polypeptide). In particular, the addition of one or more cysteine residues to the amino or carboxy-terminus of any of the polypeptides of the invention can facilitate conjugation of these polypeptides by, e.g., disulfide bonding. For example, a polypeptide can be modified to include a single cysteine residue at the amino-terminus or a single cysteine residue at the carboxy-terminus. Amino acid substitutions can be conservative (i.e., wherein a residue is replaced by another of the same general type or group) or non-conservative (i.e., wherein a residue is replaced by an amino acid of another type). In addition, a naturally occurring amino acid can be substituted for a non-naturally occurring amino acid (i.e., non-naturally occurring conservative amino acid substitution or a non-naturally occurring non-conservative amino acid substitution).

Polypeptides made synthetically can include substitutions of amino acids not naturally encoded by DNA (e.g., non-naturally occurring or unnatural amino acid). Examples of non-naturally occurring amino acids include D-amino acids, N-protected amino acids, an amino acid having an acetylaminomethyl group attached to a sulfur atom of a cysteine, a pegylated amino acid, the omega amino acids of the formula NH₂(CH₂)_(n)COOH wherein n is 2-6, neutral nonpolar amino acids, such as sarcosine, t-butyl alanine, t-butyl glycine, N-methyl isoleucine, and norleucine. Phenylglycine may substitute for Trp, Tyr, or Phe; citrulline and methionine sulfoxide are neutral nonpolar, cysteic acid is acidic, and ornithine is basic. Proline may be substituted with hydroxyproline and retain the conformation conferring properties.

Analogs may be generated by substitutional mutagenesis and retain the biological activity of the original polypeptide. Examples of substitutions identified as “conservative substitutions” are shown in Table 1. If such substitutions result in a change not desired, then other type of substitutions, denominated “exemplary substitutions” in Table 1, or as further described herein in reference to amino acid classes, are introduced and the products screened.

TABLE 1 Amino acid substitutions Original Conservative residue Exemplary substitution substitution Ala (A) Val, Leu, Ile Val Arg (R) Lys, Gln, Asn Lys Asn (N) Gln, His, Lys, Arg Gln Asp (D) Glu Glu Cys (C) Ser Ser Gln (Q) Asn Asn Glu (E) Asp Asp Gly (G) Pro Pro His (H) Asn, Gln, Lys, Arg Arg Ile (I) Leu, Val, Met, Ala, Phe, norleucine Leu Leu (L) Norleucine, Ile, Val, Met, Ala, Phe Ile Lys (K) Arg, Gln, Asn Arg Met (M) Leu, Phe, Ile Leu Phe (F) Leu, Val, Ile, Ala Leu Pro (P) Gly Gly Ser (S) Thr Thr Thr (T) Ser Ser Trp (W) Tyr Tyr Tyr (Y) Trp, Phe, Thr, Ser Phe Val (V) Ile, Leu, Met, Phe, Ala, norleucine Leu

Substantial modifications in function or immunological identity are accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.

Cross-Linking Groups

A cross-linking group is a reactive group that is able to join two or more molecules by a covalent bond. Cross-linking groups may be used to attach the linker and chelating moiety to a therapeutic or targeting moiety. Cross-linking groups may also be used to attach the linker and chelating moiety to a target in vivo. In some embodiments, the cross-linking group is an amino-reactive, methionine reactive or thiol-reactive cross-linking group, or a sortase-mediated coupling. In some embodiments, the amino-reactive or thiol-reactive cross-linking group comprises an activated ester such as a hydroxysuccinimide ester, 2,3,5,6-tetrafluorophenol ester, 4-nitrophenol ester or an imidate, anhydride, thiol, disulfide, maleimide, azide, alkyne, strained alkyne, strained alkene, halogen, sulfonate, haloacetyl, amine, hydrazide, diazirine, phosphine, tetrazine, isothiocyanate, or oxaziridine. In some embodiments, the sortase recognition sequence may comprise of a terminal glycine-glycine-glycine (GGG) and/or LPTXG (SEQ ID NO: 10) amino acid sequence, where X is any amino acid. The person having ordinary skill in the art will understand that the use of cross linking groups in the practice of the invention are not limited to the specific constructs disclosed herein, but rather may include other known cross linking groups.

Detection Agents

A detection agent is a molecule or atom which is administered conjugated to a polypeptide, e.g., an antibody or antigen-binding fragment thereof, and is useful in diagnosing a disease by locating the cells containing the antigen, radiation treatment planning, or treatment of a disease. Useful detection agents include, but are not limited to, radioisotopes, dyes (such as with the biotin-streptavidin complex), contrast agents, fluorescent compounds or molecules, luminescent agents, and enhancing agents (e.g., paramagnetic ions) for magnetic resonance imaging (MRI). In order to load a polypeptide component with a detection agent it may be necessary to react it with a reagent having a linker to which are attached the detection agent or multiple detection agents.

Radioisotopes and Radionuclides

Radioisotopes and radionuclides known in the art for their utility as detection agents include, but are not limited to, ³H, ¹⁴C, ¹⁵N, ¹⁸F, ³⁵S, ⁴⁷Sc, ⁵⁵Co, ⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ⁸⁹Zr, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y, ⁹⁷Ru, ⁹⁹Tc, ^(99m)Tc ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹In, ¹²³I, ¹²⁴I, ¹²⁵I, ¹³¹I, ¹⁴⁹Pm, ¹⁴⁹Tb, ¹⁵³Sm, ¹⁶⁶Ho, ¹⁷⁷Lu, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁹⁸Au, ¹⁹⁹Au, ²⁰³Pb, ²¹¹At, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²³Ra, ²²⁵Ac, ²²⁷Th, ²²⁹Th, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁸²Rb, ^(117m)Sn, ²⁰¹Tl.

Chelating Moieties

Chelating moieties are known in the art for their utility as detection agents include, but are not limited to, DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), DOTMA (1R,4R,7R,10R)-α, α′, α″, α′″-tetramethyl-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTAM (1,4,7,10-tetrakis(carbamoylmethyl)-1,4,7,10-tetraazacyclododecane), DOTPA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra propionic acid), DO3AM-acetic acid (2-(4,7,10-tris(2-amino-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)acetic acid), DOTA-GA anhydride (2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid, DOTP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra(methylene phosphonic acid)), DOTMP (1,4,6,10-tetraazacyclodecane-1,4,7,10-tetramethylene phosphonic acid, DOTA-4AMP (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrakis(acetamido-methylenephosphonic acid), CB-TE2A (1,4,8,11-tetraazabicyclo[6.6.2]hexadecane-4,11-diacetic acid), NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid), NOTP (1,4,7-triazacyclononane-1,4,7-tri(methylene phosphonic acid), TETPA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetrapropionic acid), TETA (1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetra acetic acid), HEHA (1,4,7,10,13,16-hexaazacyclohexadecane-1,4,7,10,13,16-hexaacetic acid), PEPA (1,4,7,10,13-pentaazacyclopentadecane-N,N′,N″,N′″, N″″-pentaacetic acid), H₄octapa (N,N′-bis(6-carboxy-2-pyridylmethyl)-ethylenediamine-N,N′-diacetic acid), H₂dedpa (1,2-[[6-(carboxy)-pyridin-2-yl]-methylamino]ethane), H₆phospa (N,N′-(methylenephosphonate)-N,N′-[6-(methoxycarbonyl)pyridin-2-yl]-methyl-1,2-diaminoethane), TTHA (triethylenetetramine-N,N,N′,N″,N′″, N′″-hexaacetic acid), DO2P (tetraazacyclododecane dimethanephosphonic acid), HP-DO3A (hydroxypropyltetraazacyclododecanetriacetic acid), EDTA (ethylenediaminetetraacetic acid), Deferoxamine, DTPA (diethylenetriaminepentaacetic acid), DTPA-BMA (diethylenetriaminepentaacetic acid-bismethylamide), HOPO (octadentate hydroxypyridinones), or porphyrins. Chelating groups may be used in metal chelate combinations with metals, such as manganese, iron, and gadolinium and isotopes (e.g., isotopes in the general energy range of 60 to 4,000 keV), such as ⁴⁷Sc, ⁵⁵Co, ⁶⁰Cu, ⁶¹Cu, ⁶²Cu, ⁶⁴Cu, ⁶⁷Cu, ⁶⁶Ga, ⁶⁷Ga, ⁶⁸Ga, ⁸²Rb, ⁸⁶Y, ⁸⁷Y, ⁹⁰Y, ⁸⁹Zr, ⁹⁷Ru, ^(99m)Tc, ¹⁰⁵Rh, ¹⁰⁹Pd, ¹¹¹In, ^(117m)Sn, ¹⁴⁹Tb, ¹⁴⁹Pm, ¹⁵³Sm, ¹⁷⁷Lu, ¹⁸⁶Re ¹⁸⁸Re, ¹⁹⁹Au, ²⁰¹Tl, ²⁰³Pb, ²¹²Pb, ²¹²Bi, ²¹³Bi, ²²⁵Ac, and ²²⁷Th.

Linkers

Linkers of the invention may have the structure of Formula I: A-L¹-(L²)_(n)-B   Formula I wherein A is chelating moiety or a metal complex thereof; L¹ is optionally substituted C₁-C₆ alkyl, substituted C₁-C₆ heteroalkyl, substituted aryl or heteroaryl; B is a is a therapeutic moiety, a targeting moiety, or cross-linking group, or a pharmaceutically acceptable salt thereof; n is 1-5; each L², independently, has the structure: (-X¹-L³-Z¹—)   Formula II wherein is X¹ is C═O(NR¹), C═S(NR¹), OC═O(NR¹), NR¹C═O(O), NR¹C═O(NR¹), CH₂PhC═O(NR¹), —CH₂Ph(NH)C═S(NR¹), O, NR¹ and R¹ is H or optionally substituted C₁-C₆ alkyl or optionally substituted C₁-C₆ heteroalkyl, substituted aryl or heteroaryl; L³ is optionally substituted C₁-C₅₀ alkyl or optionally substituted C₁-C₅₀ heteroalkyl or C₅-C₂₀ polyethylene glycol; Z¹ is CH₂, C═O, C═S, OC═O, NR¹C═O, NR¹ and R¹ is a hydrogen or optionally substituted C₁-C₆ alkyl, pyrrolidine-2,5-dione.

The conjugates of the invention comprise three distinct modules that together result in their increased effectiveness compared to those known in the art.

1. Chelating Moiety or Metal Complex Thereof:

Module A is included for incorporation of a detection agent (e.g., a chelating moiety or metal complex thereof). A metal complex may include an imaging radionuclide.

2. Linkers:

Linkers of the invention have the structure of Formula I: A-L¹-(L²)_(n)-B   Formula I

wherein A is chelating moiety or a metal complex thereof;

L¹ is optionally substituted C₁-C₆ alkyl, substituted C₁-C₆ heteroalkyl, substituted aryl or heteroaryl;

B is a is a therapeutic moiety, a targeting moiety, or cross-linking group,

or a pharmaceutically acceptable salt thereof;

n is 1-5;

each L², independently, has the structure: (-X¹-L³-Z¹-)   Formula II

wherein is X¹ is C═O(NR¹), C═S(NR¹), OC═O(NR¹), NR¹C═O(O), NR¹C═O(NR¹), —CH₂PhC═O(NR¹), —CH₂Ph(NH)C═S(NR¹), O, NR¹ and R¹ is H or optionally substituted C₁-C₆ alkyl or optionally substituted C₁-C₆ heteroalkyl, substituted aryl or heteroaryl;

L³ is optionally substituted C₁-C₅₀ alkyl or optionally substituted C₁-C₅₀ heteroalkyl or C₅-C₂₀ polyethylene glycol;

Z¹ is CH₂, C═O, C═S, OC═O, NR¹C═O, NR¹ and R¹ is a hydrogen or optionally substituted C₁-C₆ alkyl, pyrrolidine-2,5-dione.

3. Therapeutic Moiety, Targeting Moiety, or Cross-Linking Group:

Module B is a therapeutic moiety (e.g., antibodies, antigen-binding fragments), a targeting moiety (e.g. nanobodies, affibodies, consensus sequences from Fibronectin type III domains), or a cross-linking group (e.g. amino-reactive, thiol-reactive cross-linking group, or a sortase-mediated coupling). Administration and Dosage

The present invention also features pharmaceutical compositions that contain a therapeutically effective amount of a compound of the invention. The composition can be formulated for use in a variety of drug delivery systems. One or more physiologically acceptable excipients or carriers can also be included in the composition for proper formulation. Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences, Mack Publishing Company, Philadelphia, Pa., 17th ed., 1985. For a brief review of methods for drug delivery, see, e.g., Langer (Science 249:1527-1533, 1990).

The pharmaceutical compositions are intended for parenteral, intranasal, topical, oral, or local administration, such as by a transdermal means, for prophylactic and/or therapeutic treatment. The pharmaceutical compositions can be administered parenterally (e.g., by intravenous, intramuscular, or subcutaneous injection), or by oral ingestion, or by topical application or intraarticular injection at areas affected by the vascular or cancer condition. Additional routes of administration include intravascular, intra-arterial, intratumor, intraperitoneal, intraventricular, intraepidural, as well as nasal, ophthalmic, intrascleral, intraorbital, rectal, topical, or aerosol inhalation administration. Sustained release administration is also specifically included in the invention, by such means as depot injections or erodible implants or components. Thus, the invention provides compositions for parenteral administration that include the above mention agents dissolved or suspended in an acceptable carrier, preferably an aqueous carrier, e.g., water, buffered water, saline, or PBS, among others. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, wetting agents, or detergents, among others. The invention also provides compositions for oral delivery, which may contain inert ingredients such as binders or fillers for the formulation of a unit dosage form, such as a tablet or a capsule. Furthermore, this invention provides compositions for local administration, which may contain inert ingredients such as solvents or emulsifiers for the formulation of a cream, an ointment, a gel, a paste, or an eye drop.

These compositions may be sterilized by conventional sterilization techniques, or may be sterile filtered. The resulting aqueous solutions may be packaged for use as is, or lyophilized, the lyophilized preparation being combined with a sterile aqueous carrier prior to administration. The pH of the preparations typically will be between 3 and 11, more preferably between 5 and 9 or between 6 and 8, and most preferably between 6 and 7, such as 6 to 6.5. The resulting compositions in solid form may be packaged in multiple single dose units, each containing a fixed amount of the above-mentioned agent or agents, such as in a sealed package of tablets or capsules. The composition in solid form can also be packaged in a container for a flexible quantity, such as in a squeezable tube designed for a topically applicable cream or ointment.

The compositions containing an effective amount can be administered for radiation treatment planning, diagnostic, or therapeutic treatments. When administered for radiation treatment planning or diagnostic purposes, the conjugate is administered to a subject in a diagnostically effective dose and/or an amount effective to determine the therapeutically effective dose. In therapeutic applications, compositions are administered to a subject (e.g., a human) already suffering from a condition (e.g., cancer) in an amount sufficient to cure or at least partially arrest the symptoms of the disorder and its complications. An amount adequate to accomplish this purpose is defined as a “therapeutically effective amount,” an amount of a compound sufficient to substantially improve at least one symptom associated with the disease or a medical condition. For example, in the treatment of cancer, an agent or compound that decreases, prevents, delays, suppresses, or arrests any symptom of the disease or condition would be therapeutically effective. A therapeutically effective amount of an agent or compound is not required to cure a disease or condition but will provide a treatment for a disease or condition such that the onset of the disease or condition is delayed, hindered, or prevented, or the disease or condition symptoms are ameliorated, or the term of the disease or condition is changed or, for example, is less severe or recovery is accelerated in an individual. The conjugates of the invention can be used for the treatment of cancer by administering to a subject a first dose of any of the foregoing conjugates or compositions in an amount effective for radiation treatment planning, followed by administering a second dose of any of the foregoing conjugates or compositions in a therapeutically effective amount.

Amounts effective for these uses may depend on the severity of the disease or condition and the weight and general state of the subject. The therapeutically effective amount of the compositions of the invention and used in the methods of this invention applied to mammals (e.g., humans) can be determined by the ordinarily-skilled artisan with consideration of individual differences in age, weight, and the condition of the mammal. Because certain conjugates of the invention exhibit an enhanced ability to target cancer cells and residualize, the dosage of the compounds of the invention can be lower than (e.g., less than or equal to about 90%, 75%, 50%, 40%, 30%, 20%, 15%, 12%, 10%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of) the equivalent dose of required for a therapeutic effect of the unconjugated and/or non-radiolabeled agent. The agents of the invention are administered to a subject (e.g., a mammal, such as a human) in an effective amount, which is an amount that produces a desirable result in a treated subject. Therapeutically effective amounts can also be determined empirically by those of skill in the art.

Single or multiple administrations of the compositions of the invention including an effective amount can be carried out with dose levels and pattern being selected by the treating physician. The dose and administration schedule can be determined and adjusted based on the severity of the disease or condition in the subject, which may be monitored throughout the course of treatment according to the methods commonly practiced by clinicians or those described herein.

The conjugates of the present invention may be used in combination with either conventional methods of treatment or therapy or may be used separately from conventional methods of treatment or therapy.

When the compounds of this invention are administered in combination therapies with other agents, they may be administered sequentially or concurrently to an individual. Alternatively, pharmaceutical compositions according to the present invention may be comprised of a combination of a compound of the present invention in association with a pharmaceutically acceptable excipient, as described herein, and another therapeutic or prophylactic agent known in the art.

By “antiproliferative” or “antiproliferative agent,” as used interchangeably herein, is meant any anticancer agent, including those antiproliferative agents listed in Table 2, any of which can be used in combination with a conjugate of the invention to treat the medical conditions recited herein. Antiproliferative agents also include organo-platinum derivatives, naphtoquinone and benzoquinone derivatives, chrysophanic acid and anthroquinone derivatives thereof.

By “immunoregulatory agent” or “immunomodulatory agent,” as used interchangeably herein, is meant any immuno-modulator, including those listed in Table 2, any of which can be used in combination with a conjugate of the invention to treat the medical conditions recited herein.

As used herein, “radiation sensitizer” includes any agent that increases the sensitivity of cancer cells to radiation therapy. Radiation sensitizers may include, but are not limited to, 5-fluorouracil, analogs of platinum (e.g., cisplatin, carboplatin, oxaliplatin), gemcitabine, EGFR antagonists (e.g., cetuximab, gefitinib), farnesyltransferase inhibitors, COX-2 inhibitors, bFGF antagonists, and VEGF anatagonists.

TABLE 2 Alkylating agents Busulfan Chlorambucil dacarbazine procarbazine ifosfamide altretamine hexamethylmelamine estramustine phosphate thiotepa mechlorethamine dacarbazine streptozocin lomustine temozolomide cyclophosphamide Semustine Platinum agents spiroplatin lobaplatin (Aeterna) tetraplatin satraplatin (Johnson Matthey) ormaplatin BBR-3464 (Hoffmann-La Roche) iproplatin Miriplatin picoplatin AP-5280 (Access) oxaliplatin cisplatin carboplatin Antimetabolites azacytidine trimetrexate Floxuridine deoxycoformycin 2-chlorodeoxyadenosine pentostatin 6-mercaptopurine hydroxyurea 6-thioguanine decitabine (SuperGen) cytarabine clofarabine (Bioenvision) 2-fluorodeoxy cytidine irofulven (MGI Pharma) methotrexate DMDC (Hoffmann-La Roche) tomudex ethynylcytidine (Taiho) fludarabine gemcitabine raltitrexed capecitabine Topoisomerase amsacrine exatecan mesylate (Daiichi) inhibitors epirubicin quinamed (ChemGenex) etoposide gimatecan (Sigma-Tau) teniposide or mitoxantrone diflomotecan (Beaufour-Ipsen) 7-ethyl-10-hydroxy-camptothecin TAS-103 (Taiho) dexrazoxanet (TopoTarget) elsamitrucin (Spectrum) pixantrone (Novuspharma) Edotecarin rebeccamycin analogue (Exelixis) Cositecan BBR-3576 (Novuspharma) Belotecan rubitecan (SuperGen) hydroxycamptothecin (SN-38) irinotecan (CPT-11) topotecan Antitumor antibiotics valrubicin azonafide therarubicin anthrapyrazole idarubicin oxantrazole rubidazone losoxantrone plicamycin Sabarubicin porfiromycin mitoxantrone (novantrone) Epirubicin amonafide mitoxantrone doxorubicin Antimitotic colchicine E7010 (Abbott) agents vinblastine PG-TXL (Cell Therapeutics) vindesine IDN 5109 (Bayer) dolastatin 10 (NCI) A 105972 (Abbott) rhizoxin (Fujisawa) A 204197 (Abbott) mivobulin (Warner-Lambert) LU 223651 (BASF) cemadotin (BASF) D 24851 (ASTAMedica) RPR 109881A (Aventis) ER-86526 (Eisai) TXD 258 (Aventis) combretastatin A4 (BMS) epothilone B (Novartis) isohomohalichondrin-B (PharmaMar) T 900607 (Tularik) ZD 6126 (AstraZeneca) T 138067 (Tularik) AZ10992 (Asahi) cryptophycin 52 (Eli Lilly) IDN-5109 (Indena) vinflunine (Fabre) AVLB (Prescient NeuroPharma) auristatin PE (Teikoku Hormone) azaepothilone B (BMS) BMS 247550 (BMS) BNP-7787 (BioNumerik) BMS 184476 (BMS) CA-4 prodrug (OXiGENE) BMS 188797 (BMS) dolastatin-10 (NIH) taxoprexin (Protarga) CA-4 (OXiGENE) SB 408075 (GlaxoSmithKline) docetaxel Vinorelbine vincristine Trichostatin A paclitaxel Aromatase inhibitors aminoglutethimide YM-511 (Yamanouchi) atamestane (BioMedicines) formestane letrozole exemestane anastrazole Thymidylate pemetrexed (Eli Lilly) nolatrexed (Eximias) synthase inhibitors ZD-9331 (BTG) CoFactor ™ (BioKeys) DNA antagonists trabectedin (PharmaMar) edotreotide (Novartis) glufosfamide (Baxter International) mafosfamide (Baxter International) albumin + 32P (Isotope Solutions) apaziquone (Spectrum thymectacin (NewBiotics) Pharmaceuticals) O6 benzyl guanine (Paligent) Farnesyltransferase arglabin (NuOncology Labs) tipifarnib (Johnson & Johnson) inhibitors lonafarnib (Schering-Plough) perillyl alcohol (DOR BioPharma) BAY-43-9006 (Bayer) Pump inhibitors CBT-1 (CBA Pharma) zosuquidar trihydrochloride (Eli Lilly) tariquidar (Xenova) biricodar dicitrate (Vertex) MS-209 (Schering AG) Histone tacedinaline (Pfizer) pivaloyloxymethyl butyrate (Titan) acetyltransferase SAHA (Aton Pharma) depsipeptide (Fujisawa) inhibitors MS-275 (Schering AG) Metalloproteinase Neovastat (Aeterna Laboratories) CMT-3 (CollaGenex) inhibitors marimastat (British Biotech) BMS-275291 (Celltech) Ribonucleoside gallium maltolate (Titan) tezacitabine (Aventis) reductase inhibitors triapine (Vion) didox (Molecules for Health) TNF alpha virulizin (Lorus Therapeutics) revimid (Celgene) agonists/antagonists CDC-394 (Celgene) Endothelin A atrasentan (Abbott) YM-598 (Yamanouchi) receptor antagonist ZD-4054 (AstraZeneca) Retinoic acid fenretinide (Johnson & Johnson) alitretinoin (Ligand) receptor agonists LGD-1550 (Ligand) Immuno-modulators interferon dexosome therapy (Anosys) oncophage (Antigenics) pentrix (Australian Cancer GMK (Progenics) Technology) adenocarcinoma vaccine (Biomira) ISF-154 (Tragen) CTP-37 (AVI BioPharma) cancer vaccine (Intercell) IRX-2 (Immuno-Rx) norelin (Biostar) PEP-005 (Peplin Biotech) BLP-25 (Biomira) synchrovax vaccines (CTL Immuno) MGV (Progenics) melanoma vaccine (CTL Immuno) β-alethine (Dovetail) p21 RAS vaccine (GemVax) CLL therapy (Vasogen) MAGE-A3 (GSK) Ipilimumab (BMS), nivolumab (BMS) CM-10 (cCam Biotherapeutics) abatacept (BMS) atezolizumab (Genentech) pembrolizumab (Merck) Hormonal and estrogens dexamethasone antihormonal agents conjugated estrogens prednisone ethinyl estradiol methylprednisolone chlortrianisen prednisolone idenestrol aminoglutethimide hydroxyprogesterone caproate leuprolide medroxyprogesterone octreotide testosterone mitotane testosterone propionate; P-04 (Novogen) fluoxymesterone 2-methoxyestradiol (EntreMed) methyltestosterone arzoxifene (Eli Lilly) diethylstilbestrol tamoxifen megestrol toremofine bicalutamide goserelin flutamide Leuporelin nilutamide bicalutamide Photodynamic talaporfin (Light Sciences) Pd-bacteriopheophorbide (Yeda) agents Theralux (Theratechnologies) Motexafin lutetium motexafin gadolinium hypericin (Pharmacyclics) Kinase Inhibitors imatinib (Novartis) EKB-569 (Wyeth) leflunomide (Sugen/Pharmacia) kahalide F (PharmaMar) ZD1839 (AstraZeneca) CEP-701 (Cephalon) erlotinib (Oncogene Science) CEP-751 (Cephalon) canertinib (Pfizer) MLN518 (Millenium) squalamine (Genaera) PKC412 (Novartis) SU5416 (Pharmacia) Phenoxodiol (Novogen) SU6668 (Pharmacia) C225 (ImClone) ZD4190 (AstraZeneca) rhu-Mab (Genentech) ZD6474 (AstraZeneca) MDX-H210 (Medarex) vatalanib (Novartis) 2C4 (Genentech) PKI166 (Novartis) MDX-447 (Medarex) GW2016 (GlaxoSmithKline) ABX-EGF (Abgenix) EKB-509 (Wyeth) IMC-1C11 (ImClone) trastuzumab (Genentech) Tyrphostins OSI-774 (Tarceva ™) Gefitinib (Iressa) CI-1033 (Pfizer) PTK787 (Novartis) SU11248 (Pharmacia) EMD 72000 (Merck) RH3 (York Medical) Emodin Genistein Radicinol Radicinol Vemurafenib (B-Raf enzyme Met-MAb (Roche) inhibitor, Daiichi Sankyo) SR-27897 (CCK A inhibitor, Sanofi-Synthelabo) ceflatonin (apoptosis promotor, ChemGenex) tocladesine (cyclic AMP agonist, Ribapharm) BCX-1777 (PNP inhibitor, BioCryst) alvocidib (CDK inhibitor, Aventis) ranpirnase (ribonuclease stimulant, Alfacell) CV-247 (COX-2 inhibitor, Ivy Medical) galarubicin (RNA synthesis inhibitor, Dong-A) P54 (COX-2 inhibitor, Phytopharm) tirapazamine (reducing agent, SRI CapCell ™ (CYP450 stimulant, Bavarian Nordic) International) GCS-100 (gal3 antagonist, GlycoGenesys) N-acetylcysteine (reducing agent, Zambon) G17DT immunogen (gastrin inhibitor, Aphton) R-flurbiprofen (NF-kappaB inhibitor, Encore) efaproxiral (oxygenator, Allos Therapeutics) 3CPA (NF-kappaB inhibitor, Active Biotech) PI-88 (heparanase inhibitor, Progen) seocalcitol (vitamin D receptor agonist, Leo) tesmilifene (histamine antagonist, YM 131-I-TM-601 (DNA antagonist, BioSciences) TransMolecular) histamine (histamine H2 receptor agonist, Maxim) eflornithine (ODC inhibitor, ILEX Oncology) tiazofurin (IMPDH inhibitor, Ribapharm) minodronic acid (osteoclast inhibitor, cilengitide (integrin antagonist, Merck KGaA) Yamanouchi) SR-31747 (IL-1 antagonist, Sanofi-Synthelabo) indisulam (p53 stimulant, Eisai) CCI-779 (mTOR kinase inhibitor, Wyeth) aplidine (PPT inhibitor, PharmaMar) exisulind (PDE V inhibitor, Cell Pathways) gemtuzumab (CD33 antibody, Wyeth Ayerst) CP-461 (PDE V inhibitor, Cell Pathways) PG2 (hematopoiesis enhancer, AG-2037 (GART inhibitor, Pfizer) Pharmagenesis) WX-UK1 (plasminogen activator inhibitor, Wilex) Immunol ™ (triclosan oral rinse, Endo) PBI-1402 (PMN stimulant, ProMetic LifeSciences) triacetyluridine (uridine prodrug , Wellstat) bortezomib (proteasome inhibitor, Millennium) SN-4071 (sarcoma agent, Signature SRL-172 (T cell stimulant, SR Pharma) BioScience) TLK-286 (glutathione S transferase inhibitor, TransMID-107 ™ (immunotoxin, KS Biomedix) Telik) PCK-3145 (apoptosis promotor, Procyon) PT-100 (growth factor agonist, Point doranidazole (apoptosis promotor, Pola) Therapeutics) CHS-828 (cytotoxic agent, Leo) midostaurin (PKC inhibitor, Novartis) trans-retinoic acid (differentiator, NIH) bryostatin-1 (PKC stimulant, GPC Biotech) MX6 (apoptosis promotor, MAXIA) CDA-II (apoptosis promotor, Everlife) apomine (apoptosis promotor, ILEX Oncology) SDX-101 (apoptosis promotor, Salmedix) urocidin (apoptosis promotor, Bioniche) rituximab (CD20 antibody, Genentech Ro-31-7453 (apoptosis promotor, La Roche) carmustine brostallicin (apoptosis promotor, Pharmacia) Mitoxantrone β-lapachone Bleomycin gelonin Absinthin cafestol Chrysophanic acid kahweol Cesium oxides caffeic acid BRAF inhibitors, Tyrphostin AG PD-L1 inhibitors PD-1 inhibitors MEK inhibitors CTLA-4 inhibitors bevacizumab sorafenib angiogenesis inhibitors dabrafenib

The following Examples are intended to illustrate the synthesis of a representative number of conjugates and the use of these conjugates for the treatment of cancer. Accordingly, the Examples are intended to illustrate but not to limit the invention. Additional compounds not specifically exemplified may be synthesized using conventional methods in combination with the methods described herein.

EXAMPLES Example 1. General Materials and Methods

The antibodies used were HuMIgG (Aldrich, I4506) and HuMIGF-1R (AVE1642). Lutetium-177 was received from Perkin Elmer as lutetium trichloride in a 0.05 N hydrochloric acid solution, indium-111, as the trichloride salt, was obtained from Nordion, and actinium-225 was obtained as actinium-225 trinitrate from Oak Ridge National Laboratories.

Analytical HPLC-MS was performed using a Waters Acquity HPLC-MS system comprised of a Waters Acquity Binary Solvent Manager, a Waters Acquity Sample Manager (samples cooled to 10° C.), a Water Acquity Column Manager (column temperature 30° C.), a Waters Acquity Photodiode Array Detector (monitoring at 254 nm and 214 nm), a Waters Acquity TQD with electrospray ionization and a Waters Acquity BEH C18, 2.1×50 (1.7 μm) column. Preparative HPLC was performed using a Waters HPLC system comprised of a Waters 1525 Binary HPLC pump, a Waters 2489 UV/Visible Detector (monitoring at 254 nm and 214 nm) and a Waters XBridge Prep phenyl or C18 19×100 mm (5 pm) column.

HPLC elution method 1: Waters Acquity BEH C18 2.1×50 mm (1.7 pm) column; mobile phase A: H₂O (0.1% v/v TFA); mobile phase B: acetonitrile (0.1% v/v TFA); flow rate=0.3 mL/min; initial=90% A, 3-3.5 min=0% A, 4 min=90% A, 5 min=90% A.

HPLC elution method 2: Waters XBridge Prep Phenyl 19×100 mm (5 pm) column; mobile phase A: H₂O (0.1% v/v TFA); mobile phase B: acetonitrile (0.1% v/v TFA); flow rate: 10 mL/min; initial=80% A, 13 min=0% A.

HPLC elution method 3: Waters Acquity BEH C18 2.1 ×50 mm (1.7 pm) column; mobile phase A: H₂O (0.1% v/v TFA); mobile phase B: acetonitrile (0.1% v/v TFA); flow rate=0.3 mL/min; initial=90% A, 8 min=0% A, 10 min=0% A, 11 min=90% A, 12 min=90% A.

HPLC elution method 4: Waters XBridge Prep C18 OBD 19×100 mm (5 pm) column; mobile phase A: H₂O (0.1% v/v TFA); mobile phase B: acetonitrile (0.1% v/v TFA); flow rate: 10 mL/min; initial=80% A, 3 min=80% A, 13 min=20% A, 18 min=0% A.

HPLC elution method 5: Waters XBridge Prep C18 OBD 19×100 mm (5 pm) column; mobile phase A: H₂O (0.1% v/v TFA); mobile phase B: acetonitrile (0.1% v/v TFA); flow rate: 10 mL/min; initial=90% A, 3 min=90% A, 13 min=0% A, 20 min=0% A.

HPLC elution method 6: Waters XBridge Prep C18 OBD 19×100 mm (5 pm) column; mobile phase A: H₂O (0.1% v/v TFA); mobile phase B: acetonitrile (0.1% v/v TFA); flow rate: 10 mL/min; initial=75% A, 13 min=0% A, 15 min=0% A.

HPLC elution method 7: Waters XBridge Prep C18 OBD 19×100 mm (5 pm) column; mobile phase A: H₂O (0.1% v/v TFA); mobile phase B: acetonitrile (0.1% v/v TFA); flow rate: 10 mL/min; initial=80% A, 12 min=0% A, 15 min=0% A.

HPLC elution method 8: Waters XBridge Prep C18 OBD 19×100 mm (5 pm) column; mobile phase A: H₂O (0.1% v/v TFA); mobile phase B: acetonitrile (0.1% v/v TFA); flow rate: 10 mL/min; initial=90% A, 12 min=0% A, 15 min=0% A.

Analytical Size Exclusion Chromatography (SEC) was performed using a Waters system comprised of a Waters 1525 Binary HPLC pump, a Waters 2489 UV/Visible Detector (monitoring at 280 nm), a Bioscan Flow Count radiodetector (FC-3300) and TOSOH TSKgel G3000SWxl, 7.8×300 mm column. The isocratic SEC method had a flow rate=1 mL/min, with a mobile phase of 0.1M phosphate, 0.6M NaCl, 0.025% sodium azide, pH=7.

MALDI-MS (positive ion) performed using a MALDI Bruker Ultraflextreme Spectrometer.

Radio thin-layer chromatography (radioTLC) performed with Bioscan AR-2000 Imaging Scanner, carried out on iTLC-SG glass microfiber chromatography paper (Agilent Technologies, SGI0001) plates using citrate buffer (0.1 M, pH 5.5).

Example 2. Synthesis of [¹⁷⁷Lu]-Compound A-HuMIGF-1R (Commercial Standard)

The bifunctional chelating agent 2,2′,2″-(10-(2,6-dioxotetrahydro-2H-pyran-3-yl)-1,4,7,10-tetraazacyclododecane-1,4,7-triyl)triacetic acid (DOTA-GA anhydride, Compound A) was obtained from CheMatech.

The Compound A (3.0 μmoles) was dissolved in sodium acetate buffer (0.228 mL, pH 6.5). An aliquot of the Compound A solution (8 μL, 106 nmoles) was added to a solution containing the antibody HuMIGF-1R (6.7 nmoles, AVE1642) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column. The immunoconjugate (Compound A)-HuMIGF-1R was eluted from the column with acetate buffer (pH 6.5). SEC retention time: 8.2 min; MALDI-MS (positive ion): (Compound A)-HuMIGF-1R found m/z 151759; HuMIGF-1R found m/z 149835.

As a typical reaction, the Lu-177 (1.1 mCi, 14 μL) was added to a solution of Compound A-HuMIGF-1R (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1M in acetate buffer (pH 6.5)). The radiolabeling reaction was incubated at 37° C. for 30 minutes. The crude product, [¹⁷⁷Lu]-Compound A-HuMIGF-1R, was purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). SEC retention time: 8.1 min; radioTLC radiochemical purity: 99%; radiochemical yield: 74%; specific activity: 8.2 mCi/mg.

Example 3. Synthesis of 4-{[11-oxo-11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound B)

A bifunctional chelate, 4-{[11-oxo-11-(2,3,5,6-tetrafluorophenoxy)undecyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound B), was synthesized according to the scheme provided in FIG. 2. To a solution of 5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanoic acid (DOTA-GA-(tBu)₄, 50 mg, 0.07 mmol) in ACN (2.0 mL) was added DSC (50 mg, 0.21 mmol) followed by pyridine, (0.20 mL, 2.48 mmol). The reaction was stirred at room temperature for 1 hour. To the reaction mixture was added 11-aminoundecanoic acid, (70 mg, 0.36 mmol) followed by PBS solution (1.0 mL) at room temperature. The reaction was stirred for 72 hours at room temperature. The reaction mixture was filtered with syringe filter and purified directly by Prep-HPLC using method 6 to yield Intermediate 2-A (71 mg, 74.8%).

To a solution of Intermediate 2-A (40 mg, 0.03 mmol), TFP (90 mg, 0.54 mmol) and EDC (40 mg, 0.27 mmol) in ACN (1.0 mL) was added pyridine (0.05 mL, 50 mg, 0.62 mmol) at room temperature. The solution was stirred at room temperature for 24 hours. The reaction was purified directly by Prep-HPLC using method 7 to provide Intermediate 2-B (33 mg, 82.5%) as a wax after concentration using a Biotage V10 Rapid Evaporator.

Intermediate 2-B (33 mg, 0.022 mmol) was dissolved DCM/TFA (1.0 mL/2.0 mL) and allowed to stir at room temperature for 24 hours. The reaction was concentrated by air stream and purified directly by Prep-HPLC using method 8 to yield Compound B (14 mg, 50.0%) as a clear wax after concentration. An aliquot was analyzed by HPLC-MS elution method 3; retention time: 4.15 minutes; MS (positive ESI): found m/z 808.1 [M+H]⁺; C₃₆H₅₄F₄N₅O₁₁ (calc. 808.8).

¹H NMR (600 MHz, DMSO-d₆) δ 7.99-7.88 (m, 1H), 7.82 (t, J=5.5 Hz, 1H), 3.78 (broad s, 4H), 3.43 (broad s, 12H), 3.08 (broad s, 4H), 3.00 (m, 3H), 2.93 (broad s, 3H), 2.77 (t, J=7.2 Hz, 2H), 2.30 (broad s, 2H), 1.88 (broad s, 2H), 1.66 (p, J=7.3 Hz, 2H), 1.36 (m, 4H), 1.32-1.20 (m, 9H).

Example 4. Synthesis of [¹⁷⁷Lu]-Compound B-HuMIGF-1R

Compound B (0.7 μmoles) was dissolved in sodium acetate buffer (69 μL, pH 6.5). An aliquot of Compound B solution (4 μL, 40 nmoles) was added to a solution containing the antibody HuMIGF-1R (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column. The immunoconjugate Compound B-HuMIGF-1R was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound B-HuMIGF-1R: found m/z 152988 [M+H]⁺; HuMIGF-1R: found m/z 149835 [M+H]⁺.

As a typical reaction, the Lu-177 (1.15 mCi, 14 μL) was added to a solution of Compound B-HuMIGF-1R (75 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1M in acetate buffer (pH 6.5)). The radiolabeling reaction was incubated at 37° C. for 30 minutes. The crude product, [¹⁷⁷Lu]-Compound B-HuMIGF-1R, was purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). RadioTLC radiochemical purity: 99%; radiochemical yield: 75%; specific activity: 11.9 mCi/mg.

Example 5. Synthesis of 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound C)

A bifunctional chelate, 4-{[2-(2-{2-[3-oxo-3-(2,3,5,6-tetrafluorophenoxy)propoxy]ethoxy}ethoxy)ethyl]carbamoyl}-2-[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododecan-1-yl]butanoic acid (Compound C), was synthesized according to the scheme provided in FIG. 3. To a solution of 5-(tert-butoxy)-5-oxo-4-(4,7,10-tris(2-(tert-butoxy)-2-oxoethyl)-1,4,7,10-tetraazacyclododecan-1-yl)pentanoic acid (DOTA-GA(tBu)₄, 100 mg, 0.143 mmol) in ACN (8.0 mL) was added DSC (73 mg, 0.285 mmol) and pyridine (0.80 mL, 9.89 mmol). The reaction mixture was stirred for 90 min at ambient temperature. This solution was added to a semi-solution of amino-PEG3-acid (63 mg, 0.285 mmol in 1.2 mL of DMF) in a 100 mL round bottom flask. After 4 hours at ambient temperature, the reaction was worked up by concentrating to dryness under a stream of air. The crude material was purified by HPLC elution method 2 (dissolved the crude in 6 mL of 20% ACN/H₂O). The fractions containing product were pooled and concentrated under vacuum and then co-evaporated with ACN (3×2 mL). Intermediate 1-A was obtained in 82% yield.

To vial containing Intermediate 1-A (82 mg, 60 μmol) was added ACN (2 mL), NEt₃ (50 μL, 360 μmol, 6 equiv.), HBTU (23 mg, 60 μmol, 1 equiv) and a TFP solution (50 mg, 300 μmol, 5 equiv., dissolved in 250 μL of ACN). The resulting clear solution was stirred at ambient temperature for 3 hours. The reaction was worked up by concentrating the solution to dryness under an air stream and was then diluted with ACN/H₂O (1:1, 3 mL total) and purified on preparative HPLC using elution method 4. The fractions containing product were pooled and concentrated under vacuum and then co-evaporated with ACN (3×2 mL). Intermediate 1-B was obtained as a clear residue (67 mg, 74% yield).

To a vial containing Intermediate 1-B (67 mg, 64 μmol) was added DCM (2 mL) and TFA (2 mL) and the resulting solution was stirred at ambient temperature for 16 hour. Additional TFA (2 mL) was added and the reaction was stirred at ambient temperature for 6 hour. The reaction was concentrated to dryness under an air stream with the crude product being finally dissolved in ACN/H₂O (1 mL of 10% ACN/H₂O). The crude reaction solution was then purified by preparative HPLC using elution method 5. The fractions containing product were pooled, frozen and lyophilized. Compound C was obtained as a white solid (36 mg, 63% yield). An aliquot was analyzed by HPLC-MS elution method 3; retention time: 3.11 minutes; MS (positive ESI): found m/z 828.4 [M+H]⁺; C₃₄H₅₀F₄N₅O₁₄ (calc. 828.3).

¹H NMR (DMSO-d₆, 600 MHz) δ 7.97-7.91 (m, 2H), 3.77 (t, 2H, J=6.0 Hz), 3.58-3.55 (m, 2H), 3.53-3.48 (m, 8H), 3.44-3.38 (m, 10H), 3.23-3.08 (m, 11H), 3.02 (t, 2H, J=6.0 Hz), 2.93 (broad s, 4H), 2.30 (broad s, 2H), 1.87 (broad s, 2H).

Example 6. Synthesis of [¹⁷⁷Lu]-Compound C-HuMIGF-1R

The Compound C (17.5 μmoles) was dissolved in sodium acetate buffer (1.32 mL, pH 6.5). An aliquot of Compound C solution (8 μL, 91 nmoles) was added to a solution containing the antibody HuMIGF-1R (13.4 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column. The immunoconjugate Compound C-HuMIGF-1R was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound C-HuMIGF-1R found m/z 152166 [M+H]⁺; HuMIGF-1R found m/z 149724 [M+H]⁺.

As a typical reaction, the Lu-177 (1.6 mCi, 16 μL) was added to a solution of Compound C-HuMIGF-1R (150 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1M in acetate buffer (pH 6.5)). The radiolabeling reaction was incubated at ambient temperature for 20 minutes. [¹⁷⁷Lu]-Compound C-HuMIGF-1R was purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). RadioTLC radiochemical purity: 99%; radiochemical yield: 91%; specific activity: 15.6 mCi/mg.

Example 7. Saturation Binding Experiments

Saturation binding experiments measure the specific binding at equilibrium of a radioconjugate at various concentrations in order to determine the K_(d) (ligand concentration that binds to half the receptor sites at equilibrium) and B_(max) (maximum number of binding sites). In this type of binding assay, both total and nonspecific binding are measured, where specific binding to the receptor is calculated by subtracting the difference. Nonspecific binding is typically assessed by measuring radioconjugate binding in the presence of a fixed concentration of HumIGF-1R that binds to essentially all the receptors. Since all the receptors are occupied by the HumIGF-1R, the radioconjugate only binds nonspecifically. K_(d) and B_(max) values are calculated by nonlinear regression analysis and computerized curve fitting.

The purpose of this assay was to ensure that these new radioconjugates maintained binding characteristics consistent with the native antibody in an IGF-1R expressing A431 cell line. Twenty-four hours prior to the start of the experiment, 1.5×10⁵ A431 cells were seeded in 48-well microplates in 500 μl supplemented medium. The radioconjugate was diluted with binding buffer (PBS+0.5% BSA) to a range of concentrations from 0.08 nM to 40 nM; final assay concentration 0.04 to 20 nM. At the start of the assay, the media is aspirated, discarded and 500 μl of serum-free DMEM was added to each well. The plates were incubated at 37° C. for 1 hour. Following incubation, media was aspirated from each well and discarded. The cells were washed and 100 μl of binding buffer (total binding assay) or, for non-specific binding assay, 4 μM cold-antibody was added to designated wells. Plates were incubated at 4° C. for 1 hour with mild shaking. Following the blocking step, 100 μl of radioimmunoconjugate was added to each well. The plates were then incubated at 4° C. for 2 hours. Following incubation, the contents of each well was aspirated and discarded. The cells were washed twice with PBS and were then lysed with 1% Triton-X-100. The lysates were transferred to counting tubes and run with radioconjugate standards on the Wizard 1470 gamma counter to determine the radioactivity content (in counts per minute (CPM)) for each lysate. The remaining lysate from each well (25 μl) was transferred to a 96-well plate, and the protein content of each lysate determined using a standard protein quantification assay. Total, non-specific and specific ligand binding determinations, mass of bound conjugate in each lysate were calculated by converting lysate CPM to fmol bound using the specific activity of the conjugate standards and then normalizing the fmol bound to the protein content of each lysate (in milligrams). Specific binding was determined by subtracting the non-specific binding from total binding. Total, specific and non-specific binding values (fmol/mg) were plotted (y-axis) against conjugate concentration (nM, x-axis) as shown in Table 3. The K_(d) and B_(max) were derived by curve fitting of the specific binding data to a single-site hyperbola model (GraphPad Prism Software, version 7).

Results indicated that binding affinity was not changed through the changes in the linker. In addition, these changes did not alter the binding and specificity to the target.

TABLE 3 Binding Affinity Construct (K_(d)) [¹⁷⁷Lu]-Compound A-HuMIGF-1R 2.9 nM [¹⁷⁷Lu]-Compound B-HuMIGF-1R 2.0 nM [¹⁷⁷Lu]-Compound C-HuMIGF-1R 2.2 nM

Example 8. Residualization Experiments

The residualization assay was designed to determine the degree of cell retention of radioimmunoconjugate derivatives. The assay relies on the inherent ability of the IGF-1 receptor to internalize when bound to ligand and the ability to track radiolabelled compounds. In this type of binding experiment, a constant amount of radioconjugate is incubated with an IGF-1R expressing cell line for a fixed period of time. Following incubation, the cells are stripped with a mild acid buffer to remove any external or membrane-bound radioconjugate. Fresh medium is re-applied and the cells are again incubated for a pre-determined amount of time. It is during this period that cell processes degrade the radioconjugate and thereby efflux radioactive fragments back into the culture medium or retain the radioactive fragments in the cell. Residualization is determined by calculating the amount of internalized radioactivity as a percentage of the total cell-associated activity following acid wash.

A431 cells were plated in 24-well plates at a concentration of 2.5×10⁵ cells/well in full medium (DMEM). Following overnight incubation, the cells were changed to serum-free DMEM and incubated for 1 hour at 37° C. Media was decanted and plates were washed once with sterile PBS. The radioconjugate was diluted in serum-free DMEM to a concentration of 2 nM. 500 μL of radioconjugate was loaded into each well and incubated for 4 hours at 37° C. After incubation, plates were immediately placed on ice and medium was transferred into pre-labeled (non-bound) gamma counting tubes. Cells were washed once with sterile PBS, gently shaken and decanted into the (non-bound) gamma tubes. Mild acid wash buffer (pH 4.6, 500 μL) was added into all wells. Plates were incubated at 4° C. for 15 minutes and buffer was collected into pre-labeled gamma-counting tubes (membrane-bound). Warmed serum-free media (1 mL) was added to all wells and plates were incubated at 37° C. for 0 and 24 hours. Following the prescribed incubation, plates were placed on ice and processed in the following manner. Media was decanted and collected into labeled (efflux) gamma tubes. Plates were then washed once with 1 mL cold PBS and collected into efflux tubes. Acid wash buffer (pH 2.5, 500 μL) was added to all wells and plates were incubated for 5 minutes on ice. The acid wash fraction was then collected into labeled (recycled) gamma tubes. Cells were lysed with 300 μL 1% Triton X-100 for 30 minutes at room temperature. 250 μL of the cell lysate was transferred into gamma counting tubes and counted for 10 minutes. 25 μL of the cell lysate fraction was transferred to a 96-well plate for protein quantification (Pierce BCA Protein Assay). Percent residualization (FIG. 4) was determined as CPM (lysate)/CPM (efflux+recycled+lysate).

In vitro residualization experiments demonstrated that conjugation with the different linkers resulted in radioconjugates that were effectively identical in terms of cellular internalization and retention indicating that these properties of the monoclonal antibody were not altered through conjugation.

Furthermore, these data indicate that the radioimmunoconjugates are likely to undergo similar catabolic degradation after internalization into tumor cells in vivo irrespective of the appended linker structure.

Example 9. Pharmacokinetic and Metabolism Study Results for HuMIGF-1R Compounds

Groups of 4 or 5 mice (normal CD-1 or athymic CD-1 nude) were injected intravenously with approximately 15 microcuries of radiolabelled test compound. Immunoconjugates with various linkers were synthesized and radiolabelled with lutetium-177. For pharmacokinetic studies, animals were sacrificed at specific timepoints, and blood and tumor (when applicable) were analyzed for total radioactivity. For metabolism studies, animals were placed in metabolic cages (4-5 per cage) for urine and feces collection every 24 hours for up to 7 days. The radioactive content of urine and feces samples was quantified and converted to total urine or feces output based on weight. Excretion profiles for urine, feces, or total excretion (urine+feces) were generated by plotting cumulative % injected dose (% ID) over time.

The metabolic excretion profile of [¹⁷⁷Lu]-Compound B-HuMIGF-1R, and [¹⁷⁷Lu]-Compound C-HuMIGF-1R was compared was compared to [¹⁷⁷Lu]-Compound A-HuMIGF-1R. It was found that while the linker type impacted the route, rate, and extent of radioactivity excretion (FIG. 5), it did not impact to overall whole blood pharmacokinetics of the total radioactivity associated with the radioimmunoconjugate (FIG. 6). The [¹⁷⁷Lu]-Compound A-HuMIGF-1R was excreted slowly with just 13% of the injected dose (ID) eliminated over 7 days by low level urinary excretion. In contrast, excretion of the [¹⁷⁷Lu]-Compound B-HuMIGF-1R produced an increase of 210% and [¹⁷⁷Lu]-Compound C-HuMIGF-1R excretion was 310% higher. This rank order of excretion has been similar across a several antibodies tested, with Compound C producing the greatest extent of excretion. In addition, Compound B and Compound C directed distinctly different excretion routes; [¹⁷⁷Lu]-Compound B-HuMIGF-1R was eliminated predominantly through the feces and [¹⁷⁷Lu]-Compound C-HuMIGF-1R elimination was approximately equally divided between the urine and feces. This excretion pattern has also been consistent across a several biological targeting vectors tested.

Example 10. Radiotherapeutic Efficacy

Therapeutic efficacy of [²²⁵Ac]-Compound A-HuMIGF-1R, [²²⁵Ac]-Compound B-HuMIGF-1R, and [²²⁵Ac]-Compound C-HuMIGF-1R was compared to HuMIGF-1R alone and vehicle control. Therapeutic efficacy studies were carried out using the IGF-1R overexpressing colon cancer cell line Colo-205. Tumor xenografts are established in 5-7 week old female Balb/c athymic nude mice (Charles River Laboratories). Two (2) million cells mixed in 50:50 v/v in PBS and Matrigel (Becton Dickinson) were injected subcutaneously into the lower right quadrant above the thigh of each animal. Tumors were allowed to grow for 7-10 days to an initial volume of 200 mm³. Groups of tumor bearing animals (n=4-8) were injected intravenously via the lateral tail vein with 200 μL of test article. Ac-225 radiolabelled compound test articles were dosed at 20-400 nanocuries (nCi) of activity formulated in 20 mM sodium citrate pH 5.5, 0.82% NaCl, and 0.01% Tween 80. As a control, non-radiolabelled, non-conjugated antibody (HuMIGF-1R) was administered at a protein mass equivalent corresponding to the highest radioactivity dose of the actinium-225 radioimmunoconjugates tested in a study. Tumor measurements were taken 2-3 times per week with vernier calipers in two dimensions. Tumor length was defined as the longest dimension, width was measured perpendicular to the tumor length. At the same time animals were weighed. Overall body condition and general behavior were assessed daily. A typical study had a duration of 28 days. Tumor volume (mm³) was calculated from caliper measurements as an ellipsoid: Tumor growth was expressed as relative tumor volume (RTV) which is tumor volume measured on day X divided by the tumor volume measured on the day of dosing.

The therapeutic efficacy of [²²⁵Ac]-Compound A-HuMIGF-1R, [²²⁵Ac]-Compound B-HuMIGF-1R, and [²²⁵Ac]-Compound C-HuMIGF-1R was effectively equal across all compounds; with all of the actinium-225-containing radioimmunoconjugates demonstrating higher efficacy than the non-radioactive HuMIGF-1R control.

Example 11. Synthesis of [¹⁷⁷Lu]-Compound A-Human-IgG

The compound Compound A (1.34 μmoles) was dissolved in sodium acetate buffer (20 μL, pH 6.5) and added to a solution containing the antibody Human-IgG antibody (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 45 minutes at ambient temperature, the resulting immunoconjugate was purified via a HPLC SEC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). The antibody conjugate Compound A-Human-IgG. MALDI-TOF-MS (positive ion): Compound A-Human-IgG: found m/z 150360 [M+H]⁺; Human-IgG: found m/z 148339 [M+H]⁺.

As a typical reaction, the Lu-177 (1.1 mCi, 5 μL) was added to a solution of Compound A-Human-IgG (90 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5)). The radiolabeling reaction was incubated at 37° C. for 90 minutes. The crude product, [¹⁷⁷Lu]-Compound A-Human-IgG, was purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid. RadioTLC radiochemical purity: 98%; radiochemical yield: 45%; specific activity: 15.1 mCi/mg.

Example 12. Synthesis of [¹⁷⁷Lu]-Compound B-Human-IgG

Compound B (1.17 μmoles) was dissolved in sodium acetate buffer (0.117 mL, pH 6.5). An aliquot of the Compound B solution (2 μL, 10 nmoles) was added to a solution containing the antibody Human-IgG (6.7 nmoles) in a bicarbonate buffer (pH 8.5). The human IgG preparation used consisted of a purified mixture of all IgG isotypes (IgG1-4). After 1 hour at ambient temperature the antibody conjugate product was purified via a Sephadex G-50 resin packed column. Compound A-Human-IgG was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound B-Human-IgG found m/z 149949 [M+H]⁺; Human-IgG found m/z 148540 [M+H]⁺.

As a typical reaction, the Lu-177 (1.1 mCi, 5 μL) was added to a solution of Compound B-Human-IgG (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5)). The radiolabeling reaction was incubated at 37° C. for 30 minutes. The crude product, ¹⁷⁷Lu-Compound B-Human-IgG, was purified via a HPLC SEC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10 kDa,). RadioTLC radiochemical purity: 98%; radiochemical yield: 51%; specific activity: 9.68 mCi/mg.

Example 13. Synthesis of [¹⁷⁷Lu]-Compound C-Human-IgG

The compound Compound C (0.96 μmoles) was dissolved in sodium acetate buffer (95 μL, pH 6.5). An aliquot of the Compound C solution (2 μL, 20 nmoles) was added to a solution containing the antibody Human-IgG antibody (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate product was purified via a Sephadex G-50 resin packed column. Compound C-Human-IgG was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound C-Human-IgG: found m/z 150095 [M+H]⁺; Human-IgG: found m/z 148540 [M+H]⁺.

As a typical reaction, the Lu-177 (1.1 mCi, 5 μL) was added to a solution of Compound C-Human-IgG (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5)). The radiolabeling reaction was incubated at 37° C. for 30 minutes. The crude product, [¹⁷⁷Lu]-Compound C-Human-IgG, was purified via a HPLC SEC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10 kDa,). RadioTLC radiochemical purity: 98%; radiochemical yield: 37%; specific activity: 9.99 mCi/mg.

Example 14. Pharmacokinetic and Metabolism Study Results for HuMIgG Based Compounds

Non-targeted human IgG antibodies were used for metabolic excretion studies in order to demonstrate that the alterations in radioactivity excretion profiles directed by conjugation with linker Compound B and Compound C is a general process demonstrating that these finding are not-limited to HuMIGF-1R antibody. Pharmacokinetic and metabolism studies were carried out using [¹⁷⁷Lu]-Compound A-HuMIgG, [¹⁷⁷Lu]-Compound B-HuMIgG, and [¹⁷⁷Lu]-Compound C-HuMIgG as described for the HuMIGF-1R antibody based compounds described previously.

The metabolic excretion profile of a non-targeted human IgG radioimmunoconjugates [¹⁷⁷Lu]-Compound B-HuMIgG, and [¹⁷⁷Lu]-Compound C-HuMIgG were compared to [¹⁷⁷Lu]-Compound A-HuMIgG. As described for the HuMIGF-1R-based radioimmunoconjugates, it was found that while the linker type impacted the route, rate, and extent of radioactivity excretion (FIG. 8), it did not impact the overall pharmacokinetics of the total radioactivity associated with the radioimmunoconjugate. The same rank order of excretion was observed for the HuMIgG-based compounds as was seen for the HuMIGF-IR based compounds; that is Compound C-containing radioimmunoconjugate producing the greatest extent of excretion. The [¹⁷⁷Lu]-Compound A-HuMIgG was excreted slowly with just 13% of the injected dose (ID) eliminated over 7 days by low level urinary excretion. In contrast, excretion of the [¹⁷⁷Lu]-Compound B-HuMIGF-1R gave an increase of 196% and [¹⁷⁷Lu]-Compound C-HuMIGF-1R excretion was 216% higher. In addition, Compound B and Compound C directed distinctly different excretion routes. [¹⁷⁷Lu]-Compound B-HuMIgG was predominantly eliminated through the feces whereas [¹⁷⁷Lu]-Compound C-HuMIgG elimination was roughly equally divided between the urine and feces. This metabolic profile was fundamentally equivalent for the HuMIGF-1R based compounds demonstrating that improved excretion profile of the Compound B or Compound C, when conjugated to antibodies, is a general and reproducible effect.

Example 15: Synthesis of [²²⁵Ac]-Compound A-Human-IgG

The compound Compound A (1.34 μmoles) was dissolved in sodium acetate buffer (20 μL, pH 6.5) and added to a solution containing the antibody Human-IgG antibody (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 45 minutes at ambient temperature the antibody conjugate product was purified via a HPLC SEC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid). MALDI-TOF-MS (positive ion): Compound A-Human-IgG: found m/z 150360 [M+H]⁺; Human-IgG: found m/z 148339 [M+H]⁺.

As a typical reaction, Ac-225 (1.1 mCi, 5 μL) is added to a solution of Compound A-Human-IgG (90 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5)). The radiolabeling reaction is incubated at ambient temperature (e.g., 20-25° C.) for 90 minutes. The crude product, [²²⁵Ac]-Compound A-Human-IgG, is purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid).

Example 16. Synthesis of [²²⁵Ac]-Compound B-Human-IgG

The compound Compound B (1.17 μmoles) was dissolved in sodium acetate buffer (0.117 mL, pH 6.5). An aliquot of the Compound B solution (2 μL, 10 nmoles) was added to a solution containing the antibody Human-IgG (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature the antibody conjugate product was purified via a Sephadex G-50 resin packed column. The antibody conjugate Compound A-Human-IgG was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound B-Human-IgG found m/z 149949 [M+H]⁺; Human-IgG found m/z 148540 [M+H]⁺.

As a typical reaction, the Ac-225 (1.1 mCi, 5 μL) is added to a solution of Compound B-Human-IgG (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5)). The radiolabeling reaction is incubated at ambient temperature (e.g., 20-25° C.) for 30 minutes. The crude product, [²²⁵Ac]-Compound B-Human-IgG, is purified via a HPLC SEC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10 kDa).

Example 17. Synthesis of [²²⁵Ac]-Compound C-Human-IgG

The compound Compound C (0.96 μmoles) was dissolved in sodium acetate buffer (95 μL, pH 6.5). An aliquot of the Compound C solution (2 μL, 20 nmoles) was added to a solution containing the antibody Human-IgG antibody (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature the antibody conjugate product was purified via a Sephadex G-50 resin packed column. The antibody conjugate Compound C-Human-IgG was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound C-Human-IgG: found m/z 150095 [M+H]⁺; Human-IgG: found m/z 148540 [M+H]⁺.

As a typical reaction, the Ac-225 (1.1 mCi, 5 μL) is added to a solution of Compound C-Human-IgG (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5)). The radiolabeling reaction is incubated at ambient temperature (e.g., 20-25° C.) for 30 minutes. The crude product, [²²⁵Ac]-Compound C-Human-IgG, is purified via a HPLC SEC column (1 mL/min, eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid) and concentrated by ultrafiltration (Vivaspin, 10 kDa,).

Example 18. Synthesis of [²²⁵Ac]-Compound A-HuMIGF-1R

The Compound A (3.0 μmoles) was dissolved in sodium acetate buffer (0.228 mL, pH 6.5). An aliquot of the Compound A solution (8 μL, 106 nmoles) was added to a solution containing the antibody HuMIGF-1R (6.7 nmoles, AVE1642) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column. The immunoconjugate (Compound A)-HuMIGF-1R was eluted from the column with acetate buffer (pH 6.5). SEC retention time: 8.2 min; MALDI-MS (positive ion): (Compound A)-HuMIGF-1R found m/z 151759; HuMIGF-1R found m/z 149835.

As a typical reaction, the Ac-225 (1.1 mCi, 14 μL) is added to a solution of (Compound A)-HuMIGF-1R (100 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5)). The radiolabeling reaction is incubated at ambient temperature (e.g., 20-25° C.) for 30 minutes. The crude product, [²²⁵Ac]-Compound A-HuMIGF-1R, is purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid).

Example 19. Synthesis of [²²⁵Ac]-Compound B-HuMIGF-1R

Compound B (0.7 μmoles) was dissolved in sodium acetate buffer (69 μL, pH 6.5). An aliquot of Compound B solution (4 μL, 40 nmoles) was added to a solution containing the antibody HuMIGF-1R (6.7 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column. The immunoconjugate Compound B-HuMIGF-1R was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound B-HuMIGF-1R: found m/z 152988 [M+H]⁺; HuMIGF-1R: found m/z 149835 [M+H]⁺.

As a typical reaction, the Ac-225 (1.15 mCi, 14 μL) is added to a solution of Compound B-HuMIGF-1R (75 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5)). The radiolabeling reaction is incubated at ambient temperature (e.g., 20-25° C.) for 30 minutes. The crude product, [²²⁵Ac]-Compound C-HuMIGF-1R, is purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid).

Example 20. Synthesis of [²²⁵Ac]-Compound C-HuMIGF-1R

The Compound C (17.5 μmoles) was dissolved in sodium acetate buffer (1.32 mL, pH 6.5). An aliquot of Compound C solution (8 μL, 91 nmoles) was added to a solution containing the antibody HuMIGF-1R (13.4 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column. The immunoconjugate Compound C-HuMIGF-1R was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound C-HuMIGF-1R found m/z 152166 [M+H]⁺; HuMIGF-1R found m/z 149724 [M+H]⁺.

As a typical reaction, the Ac-225 (1.6 mCi, 16 μL) is added to a solution of Compound C-HuMIGF-1R (150 μg in acetate buffer (pH 6.5) and ascorbic acid (1 μL, 0.1 M in acetate buffer (pH 6.5)). The radiolabeling reaction is incubated at ambient temperature (e.g., 20-25° C.) for 30 minutes. [²²⁵Ac]-Compound C-HuMIGF-1R is purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid).

Example 21. Synthesis of [¹¹¹n]-Compound C-HuMIGF-1R

Compound C (17.5 μmoles) was dissolved in sodium acetate buffer (1.32 mL, pH 6.5). An aliquot of Compound C solution (8 μL, 91 nmoles) was added to a solution containing the antibody HuMIGF-1R (13.4 nmoles) in a bicarbonate buffer (pH 8.5). After 1 hour at ambient temperature, the resulting immunoconjugate was purified via a Sephadex G-50 resin packed column. The immunoconjugate Compound C-HuMIGF-1R was eluted from the column with acetate buffer (pH 6.5). MALDI-TOF-MS (positive ion): Compound C-HuMIGF-1R found m/z 152166 [M+H]+; HuMIGF-1R found m/z 149724 [M+H]+.

As a typical reaction, the In-111 (60 mCi, 215 μL) is added to a solution of Compound C HuMIGF-1R (7 mg, 1.6 mL in acetate buffer (pH 6.5)) and ascorbic acid (100 μL, 0.2 M in acetate buffer (pH 6.5)). The radiolabeling reaction is incubated at 37° C. for 30 minutes. [¹¹¹In]-Compound C-HuMIGF-1R is purified via a Sephadex G-50 resin packed column eluted with acetate buffer (pH 6.5, 1 mM ascorbic acid).

Example 22. In Vitro Potency of Compound C-HuMIGF-1R Conjugates

The in vitro potency of Compound C-HuMIGF-1R conjugates were measured in an assay based on the competitive inhibition of IGF-1 ligand stimulated autophosphorylation of IGF-1R using the human A431 epidermoid carcinoma cell culture model. The purpose of this set of experiments was to assess the functional impact of conjugation of antibody HuMIGF-1R to form Compound C-HuMIGF-1R.

The potency assay was performed as follows: twenty-four hours prior to the start of the experiment approximately 5×10⁴ A431 cells were seeded in 96-well microplates in growth medium and grown overnight a 37° C. The following day, Compound C-HuMIGF-1R or HuMIGF-1R were diluted with growth medium to a range of concentrations from 0.005 to 2.4 μg/mL; final assay concentration 0.004 to 2 μg/mL. At the start of the assay, the growth media was aspirated, discarded and 100 μl of the diluted compound preparations were added to the plate wells. The plates were incubated at 37° C. for 1 h. Following incubation, cells were stimulated by the addition of 20 μl of 100 ng/mL IGF-1 ligand (R&D Systems, #291-G1) for 10 minutes at room temperature. Cells were then washed with PBS buffer and lysed in a detergent containing buffer (1% IGEPAL CA-630, 20 mM Tris (pH 8.0), 137 mM NaCl). Phosphorylated IGF-IR content of the lysate was quantified using a commercially available ELISA assay (R&D Systems, #DYC-1770) according to the manufacturers' protocol. ELISA data was analyzed using Graphpad Prism software (version 7) to generate IC₅₀ (half-maximal (50%) inhibitory concentration) values for each compound tested.

Compound C-HuMIGF-1R batches were prepared with different degrees of substitution. The degree of substitution was determined using MALDI-TOF and ranged from 1.9-7.4 DOTA chelates per mAb (referred herein as the average number of DOTA per mAb or DOTA/mAb). The Compound C-HuMIGF-1R were tested for inhibitory function (measured as an IC₅₀) and were compared with unmodified antibody HuMIGF-1R in the assay (Table 4). Conjugation of antibody HuMIGF-1R was found to have minimal impact on its ability to inhibit ligand induced autophosphorylation of IGF-1R in vitro even at high degrees of substitution (up to 7.4 DOTA/mAb). Based on this data all subsequent biological studies were carried out using immunoconjugates with a degree of substitution between 3.5 and 6.5 DOTA/mAb.

The potency assay allows for direct comparison of Compound C-HuMIGF-1R with parental antibody HuMIGF-1R, and it also can be used to compare the inhibitory function of corresponding radiolabeled compounds. Compound C-HuMIGF-1R can be radiolabelled with different radiometals to suit the required application. [¹⁷⁷Lu]-Compound C-HuMIGF-1R can be used for in vitro bioassays and non-clinical biodistribution studies, [¹¹¹In]-Compound C-HuMIGF-1R can be used for in vivo imaging applications, and [²²⁵Ac]-Compound C-HuMIGF-1R can be used in radiotherapeutic applications. The purpose of this set of experiments was to assess the functional impact of radiolabeling Compound C-HuMIGF-1R conjugates with different isotopes.

Testing of [²²⁵Ac]-Compound C-HuMIGF-1R, [¹¹¹In]-Compound C-HuMIGF-1R, and [¹⁷⁷Lu]-Compound C-HuMIGF-1R revealed that radiolabeling has minimal impact on the ability of Compound C-HuMIGF-1R to inhibit IGF-1R autophosphorylation (Table 5) and demonstrates the functional equivalence of the radiolabeled compounds irrespective of the isotope used.

TABLE 4 Potency of Compound C-HuMIGF-1R at various degrees of substitution Degree of Substitution Conjugate Potency (DOTA/mAb) (IC₅₀; μg/mL) 0^(a) 0.04 1.9 0.05 3.8 0.07 4.9 0.10 6.4 0.13 7.4 0.14 ^(a)Native antibody, HuMIGF-1R

TABLE 5 Comparison of HuMIGF-1R, Compound C-HuMIGF-1R, [²²⁵Ac]-Compound C-HuMIGF-1R, [¹¹¹In]-Compound C-HuMIGF-1R, and [¹⁷⁷Lu]-Compound C-HuMIGF-1R in the in vitro potency assay Compound Potency Compound (IC₅₀; μg/mL) HuMIGF- 1R 0.04 Compound C-HuMIGF-1 R 0.09 [²²⁵Ac]-Compound C-HuMIGF-1R 0.13 [¹¹¹In]-Compound C-HuMIGF-1R 0.19 [¹⁷⁷Lu]-Compound C-HuMIGF-1R 0.04

Example 23. Saturation Binding Experiments Using a Variety of IGF-1R Expressing Human Cancer Cells Lines

Saturation binding experiments measure the specific binding at equilibrium of a radioligand at various concentrations to determine the K_(d) (ligand concentration that binds to half the receptor sites at equilibrium) and B_(max) (maximum number of binding sites). In this type of binding assay, both total and nonspecific binding are measured, where specific binding to the receptor is calculated by subtracting the difference. Nonspecific binding is typically assessed by measuring radioligand binding in the presence of a fixed concentration of an unlabeled compound that binds to essentially all the receptors. Since all the receptors are occupied by the unlabeled drug, the radioligand only binds nonspecifically. K_(d) and B_(max) values are calculated by nonlinear regression analysis and computerized curve fitting.

The purpose of this set of experiments was to demonstrate that [¹¹¹In]-Compound C-HuMIGF-1R and [¹⁷⁷Lu]-Compound C-HuMIGF-1R maintained binding characteristics consistent with the native antibody and demonstrate compound binding to a number of IGF-1R expressing cell lines representing non-small cell lung cancer (NSCLC), prostate, and pancreatic cancers. Twenty-four hours prior to the start of the experiment, 1.5×10⁵ cells were seeded in 48-well microplates in 500 μl supplemented medium. The radiolabeled conjugate was diluted with binding buffer (PBS+0.5% BSA) to a range of concentrations from 0.08 nM to 40 nM; final assay concentration 0.04 to 20 nM. At the start of the assay, the media is aspirated, discarded and 500 μl of serum-free DMEM was added to each well. The plates were incubated at 37° C. for 1 h. Following incubation, media was aspirated from each well and discarded. The cells were washed and 100 μl of binding buffer (total binding) or 4 μM cold-antibody (non-specific binding) added to designated wells. Plates were incubated at 4° C. for 1 h with mild shaking. Following the blocking step, 100 μl of labeled conjugate was added to each well. The plates were then incubated at 4° C. for 2 h. Following incubation, the contents of each well was aspirated and discarded. The cells were washed twice with PBS and were then lysed with 1% Triton-X-100. The lysates were transferred to counting tubes and run with labeled conjugate standards on the Wizard 1470 gamma counter to determine the radioactivity content (in counts per minute (CPM)) for each lysate. The remaining lysate from each well (25 μl) was transferred to a 96-well plate, and the protein content of each lysate determined using a standard protein quantification assay. Total, non-specific and specific ligand binding determinations, mass of bound conjugate in each lysate were calculated by converting lysate CPM to fmol bound using the specific activity of the conjugate standards and then normalizing the fmol bound to the protein content of each lysate (in milligrams). Specific binding was determined by subtracting the non-specific binding from total binding. Total, specific and non-specific binding values (fmol/mg) were plotted (y-axis) against conjugate concentration (nM, x-axis). The K_(d) and B_(max) were derived by curve fitting of the specific binding data to a single-site hyperbola model (GraphPad Prism Software, version 7).

The binding affinity of [¹¹¹In]-Compound C-HuMIGF-1R and [¹⁷⁷Lu]-Compound C-HuMIGF-1R to IGF-1R target was measured in vitro using the A431 epidermoid carcinoma cell culture model; a model in which IGF-1R is highly expressed. The binding affinity (or K_(d)) of [¹¹¹In]-Compound C-HuMIGF-1R and [¹⁷⁷Lu]-Compound C-HuMIGF-1R was measured to be 6.9 and 2.2 nM, respectively. This demonstrates high affinity for binding to IGF-1R expressed on tumor cells and that the binding affinity was similar irrespective of the radionuclide used for radiolabeling. Unmodified antibody HuMIGF-1R has been previously measured to have a binding affinity (K_(d)) of 0.21 nM using recombinant IGF-1R in an ELISA based assay. HuMIGF-1R binds IGF-1R from cynomolgus monkey peripheral blood cell lysates (K_(d)=0.36 nM) with an affinity similar to human IGF-1R (HuT-78 cells; K_(d)=0.21 nM). See, for example, Tavares D. Humanization of the murine anti-Igf1 receptor antibody by variable region resurfacing, Preclinical Report IMM16-001 (2006); and Maloney E. K. The binding of AVE1642 antibody to IGF-1R receptor of several species, Preclinical Draft Report IMB16-001 (2006).

Binding studies were also performed using a panel of human cancer cell lines expressing various levels of IGF-1R. [¹⁷⁷Lu]-Compound C-HuMIGF-1R was chosen for these in vitro studies based on its facile radiolabeling conditions and ease of quantification. All cell lines tested demonstrated high affinity binding of [¹⁷⁷Lu]-Compound C-HuMIGF-1R with K_(d) measured in the low nM range (Table 6). In addition, the B_(max) binding parameter, a theoretical value of target levels calculated by curve fitting which estimates the maximum level of target sites available for radioimmunoconjugate binding, agreed well with expression levels observed for the various cell lines by immunoblotting. These data demonstrated that the antibody HuMIGF-1R based radioimmunoconjugate [¹⁷⁷Lu]-Compound C-HuMIGF-1R is capable of binding IGF-1R target on a variety of different cancer cell types in vitro and that binding is approximately proportional to the amount of target expressed.

TABLE 6 In vitro binding characteristics of [¹⁷⁷Lu]-Compound C-HuMIGF-1R on IGF-1R expressing cell lines. Cancer K_(d) B_(max) Relative Expression Type Cell Line (nM) (fmol/mg) (via Immunoblot) Epidermoid A431 2.2 140.5 High Carcinoma NSCLC H226 2.3 85.5 Intermediate A549 0.7 94.8 High Pancreatic MiaPaca-II 1.9 68.7 Intermediate BxPC3 2.9 50.6 Intermediate Prostate PC-3 1.7 16.9 Low Du-145 3.0 88.4 Intermediate LnCaP 1.6 36.1 Low

Example 24. Biodistribution of Radiolabeled HuMIGF-1R Compounds

The ability of [²²⁵Ac]-Compound C-HuMIGF-1R, [¹⁷⁷Lu]-Compound C-HuMIGF-1R, and [¹¹¹In]-Compound C-HuMIGF-1R to target antigen expressing human tumors in vivo was demonstrated using the Colo-205 colorectal adenocarcinoma xenograft model. Colo-205 tumor xenografts (2 million cells per animal in Matrigel) were established female Balb/c athymic nude mice and allowed to grow for 7 days before initiation of biodistribution studies.

Animals were injected intravenously via the lateral tail vein with 0.2 mL of [²²⁵Ac]-Compound C-HuMIGF-1R test article containing approximately 0.4 μCi of radioactivity (7.8 μg of antibody) formulated in 20 mM sodium citrate pH 5.5, 0.82% NaCl, and 0.01% Tween 80. After blood collection via cardiac puncture and sacrifice at 4, 24, 48, 96, 168 hours post-injection (p.i.) (n=3 mice/timepoint), organs and tissue samples were obtained by dissection. Radioactivity measurements using a liquid scintillation counter following tissue digestion, and sample weights were used to calculate the percent of injected dose per gram of tissue weight (% ID/g).

[²²⁵Ac]-Compound C-HuMIGF-1R biodistribution data (FIG. 9) demonstrated high levels of uptake in Colo205 xenografts reaching 10.4±3.0% ID/g after 24 h and peaking at 36.2±4.5% ID/g after 168 h. The tumor contained the highest concentration (% ID/g) of radioactivity of all tissues/organs isolated across all timepoints followed by the blood. Blood clearance of [²²⁵Ac]-Compound C-HuMIGF-1R declined in a biphasic manner from 39.8±0.3% ID/g at 4 hours p.i. to 14.4±0.4% ID/g at 168 hours p.i. Highly perfused organs such as the liver, kidney, spleen and lungs showed low compound uptake and demonstrated an organ-dependent clearance pattern which was consistent with blood clearance.

This finding also suggested that the majority of radioactivity contained in these organs was due to blood perfusion rather than off-target binding.

The biodistribution of [¹⁷⁷Lu]-Compound C-HuMIGF-1R and [¹¹¹In]-Compound C-HuMIGF-1R (FIGS. 10 and 11) were also tested in the Colo-205 xenograft model. Animals were injected intravenously via the lateral tail vein with 0.2 mL of test article formulated in 20 mM sodium citrate pH 5.5, 0.82% NaCl, and 0.01% Tween 80. In the case of [¹⁷⁷Lu]-Compound C-HuMIGF-1R, 11-16 μCi of radioactivity (1 μg of antibody) were administered per animal. In the case of [¹¹¹In]-Compound C-HuMIGF-1R, 15-18 μCi of radioactivity (1 μg of antibody) were administered per animal. After blood collection via cardiac puncture and sacrifice at 4, 24, 48, 96, 168 hours post-injection (p.i.) (n=3 mice/timepoint), organs and tissue samples were obtained by dissection. Radioactivity measurements using a gamma counter and sample weights were used to calculate the percent of injected dose per gram of tissue weight (% ID/g).

The results for both [¹⁷⁷Lu]-Compound C-HuMIGF-1R and [¹¹¹In]-Compound C-HuMIGF-1R were very similar in terms of tumor uptake, blood clearance and tissue/organ uptake and were also both effectively equivalent to [²²⁵Ac]-Compound C-HuMIGF-1R. Peak tumor uptake for [¹⁷⁷Lu]-Compound C-HuMIGF-1R was 45.3±6.7% ID/g after 168 h and blood levels declined from 36.1±1.7 at 4 hours, p.i. to 12.7±2.2 at 168 hours, p.i. Likewise, peak tumor uptake for [¹¹¹In]-Compound C-HuMIGF-1R was 50.7±8.7% ID/g after 168 h and blood levels declined from 32.1±4.6% ID/g at 4 hours, p.i., to 13.8±2.2% ID/g at 168 hour, p.i.

In summary, the biodistribution of the HuMIGF-1R derived radioimmunoconjugates, [²²⁵Ac]-Compound C-HuMIGF-1R, [¹¹¹In]-Compound C-HuMIGF-1R, and [¹⁷⁷Lu]-Compound C-HuMIGF-1R, in Colo-205 xenograft bearing mice of were found to be equivalent in terms of tumor uptake, blood clearance, and tissue/organ uptake irrespective of the radionuclide used for radiolabeling. Furthermore, this indicates that [¹¹¹In]-Compound C-HuMIGF-1R is a suitable surrogate of [²²⁵Ac]-Compound C-HuMIGF-1R for use in pre-clinical or clinical imaging studies.

Example 25. Blocking Biodistribution of [¹⁷⁷Lu]-Compound C-HuMIGF-1R

Target specificity of [¹⁷⁷Lu]-Compound C-HuMIGF-1R with respect to antibody HuMIGF-1R was investigated in a biodistribution study which included the co-administration of antibody HuMIGF-1R “block” at increasing doses. Study animals bearing Colo-205 tumor xenografts were given an [¹⁷⁷Lu]-Compound C-HuMIGF-1R dose of approximately 15 μCi of radioactivity (˜1 μg of antibody) along with up to 800 μg of antibody HuMIGF-1R (n=4 animals/dose level). The blocking dose of antibody HuMIGF-1R used was in a 0-800 fold molar excess compared to [¹⁷⁷Lu]-Compound C-HuMIGF-1R. Animals were euthanized at 96 h p.i. organs and tissue samples were obtained by dissection. Radioactivity measurements using a gamma counter and sample weights were used to calculate the percent of injected dose per gram of tissue weight (% ID/g) (FIG. 12).

Blocking of tumor uptake was achieved by administration of excess antibody HuMIGF-1R in conjunction with [¹⁷⁷Lu]-Compound C-HuMIGF-1R. Antibody HuMIGF-1R had no effect on [¹⁷⁷Lu]-Compound C-HuMIGF-1R uptake in all other organs/tissues demonstrating that off tumor uptake in mice was not target dependent and confirming that [¹⁷⁷Lu]-Compound C-HuMIGF-1R binds specifically to the same tumor site in vivo as does antibody HuMIGF-1R.

Example 26. Single Dose Radiotherapeutic Efficacy of [²²⁵Ac]-Compound C-HuMIGF-1R in Colorectal, Lung and Prostate Tumor Xenograft Bearing Mice

The radiotherapeutic efficacy of [²²⁵Ac]-Compound C-HuMIGF-1R was tested using mouse models bearing xenografts of prostate (LNCaP), radioresistant lung (A459) or colorectal cancer (Colo-205). Tumor xenografts were established (2 million cells per animal in Matrigel) in female Balb/c athymic nude mice (male NCR nude mice for LNCaP xenografts) and allowed to grow for a minimum of 7 days before initiation of therapy studies. Unless otherwise noted, doses were injected intravenously via the lateral tail vein with 100-200 μL of test article (n=5 animals/group) 20 mM sodium citrate pH 5.5, 0.82% NaCl, and 0.01% Tween 80. Actinium-225 radiolabeled compound test articles were dosed at 0.05-0.4 μCi of activity (up to 10 μg of antibody depending on specific activity for a given study). Tumor measurements, in two dimensions, were taken (in mm) two to three times per week with Vernier calipers and animals were weighed prior to each measurement. Overall body condition and general behavior were assessed daily. A typical study had a duration of approximately 28 days although some studies monitored efficacy for extended periods of time (>150 days) to determine long-term efficacy and survival (Kaplan-Meier analysis) after treatment. Tumor growth was expressed as relative tumor volume (RTV), which is the tumor volume measured on a specific day divided by the tumor volume measured on the day of dosing. Tumor suppression is defined as a reduction in growth compared to control groups. Tumor regression is defined as a reduction of the tumor volume (shrinkage) compared to its initial volume at the time of dosing. Statistical significance of differences in tumor volume between compound treated animals and vehicle treated animals were analysed by ANOVA using Dunnett's multiple comparison and p-values are included on the graphs (non-significant differences indicated by “n.s.”). RTV data is plotted versus time using “last observation carried forward” when animals reached study endpoints and were euthanized over the course of the study. Study endpoints included loss of body mass (>20%), deterioration of body condition, signs of pain or altered behavior, growth of large tumors (>10% of body mass estimated using a tumor density of 1 g/cm³; loss of mobility), and tumor ulceration.

Radiotherapeutic studies of [²²⁵Ac]-Compound C-HuMIGF-1R in colorectal (Colo-205), prostate (LNCaP), and radioresistant lung (A549) cancer xenograft models demonstrated single dose radiotherapeutic efficacy (FIGS. 13-15). Groups of xenograft bearing animals (n=5/group; 200 mm³ tumors) were given single doses of [²²⁵Ac]-Compound C-HuMIGF-1R at 0.05, 0.2 and 0.4 μCi per animal. The antibody protein doses were 2.9, 8.0, and 7.2 μg for the Colo-205, A549, and LNCaP bearing animals respectively. Groups of control mice were dosed with vehicle and tumor growth was monitored over 28-29 days. Regression Colo-205 tumors was observed at the 0.2 and 0.4 μCi doses, with tumor suppression at the 0.05 μCi dose. Regression of LNCaP tumours was observed at the 0.4 μCi dose, with tumor suppression at the 0.05 and 0.2 μCi doses. Growth suppression of radioresistant A549 tumours was observed at all doses administered.

Tumor regression was also observed using 0.2 and 0.4 μCi doses in a separate experiment where colorectal (Colo-205) xenograft tumors (n=5/group) were allowed to grow to starting volume of greater than 400 mm³ prior to dosing demonstrating that [²²⁵Ac]-Compound C-HuMIGF-1R was also effective against large tumors (FIG. 16). Unconjugated (native) antibody HuMIGF-1R had no single dose efficacy at antibody protein doses of up to 800 μg (40 mg/kg) in the Colo-205 model which is at least 100-fold higher than the antibody mass in efficacious radiotherapeutic doses (FIG. 17). Furthermore, unconjugated (native) antibody HuMIGF-1R had no single dose efficacy at antibody protein doses of up to 800 μg (40 mg/kg) in the radioresistant lung (A549) model (FIG. 18).

In summary, data generated in pre-clinical models demonstrate a high degree of anti-tumor efficacy mediated by single-dose [²²⁵Ac]-Compound C-HuMIGF-1R radiotherapy in multiple tumor xenograft types. In addition, these data demonstrate targeted alpha-therapy delivered by can be effective against cell lines resistant to ionizing radiation (e.g., A549).

Example 27. Extended Single Dose Radiotherapeutic Efficacy of [²²⁵Ac] Compound C-HuMIGF-1R in Colorectal Tumor Xenograft Bearing Mice

The long-term extended efficacy of [²²⁵Ac]-Compound C-HuMIGF-1R was demonstrated in colorectal cancer (Colo-205) xenograft bearing animals (n=5/group) receiving a single dose of [²²⁵Ac]-Compound C-HuMIGF-1R at 0.05, 0.2 and 0.4 μCi per animal (2.9 μg of antibody protein). Groups of control mice were dosed with vehicle and tumor growth was monitored over 178 days as described for the radiotherapy studies detailed above. [²²⁵Ac]-Compound C-HuMIGF-1R demonstrated suppression of tumor growth at the 0.05 μCi dose and tumor regression in the 0.2 and 0.4 μCi dose groups which continued for the duration of the study period (FIG. 19). Treatment was well tolerated at all doses. The control group had a median survival of 42 days post treatment while median survival for the 0.05 and 0.4 μCi dose groups were 66 and 151 days respectively. Median survival of the 0.4 μCi dose group remained undefined at study termination. These differences in survival were statistically significant (p<0.0001, Mantel-Cox test) (FIG. 20).

Example 28. Fractionated Multi-Dose Radiotherapeutic Efficacy of [²²⁵Ac]-Compound C-HuMIGF-1R in Colorectal and Lung Tumor Xenograft Bearing Mice

The radiotherapeutic efficacy of [²²⁵Ac]-Compound C-HuMIGF-1R was tested using mouse models bearing xenografts radioresistant lung (A459) or colorectal cancer (Colo-205). Instead of treating the animals with a single dose of compound, multiple fractionated doses were administered to compare the efficacy of the different dosing regimens. Aside from the change in dosing regimens, the radiotherapy studies were carried out as previously described above.

Mice bearing colorectal (Colo-205) tumor xenografts were administered [²²⁵Ac]-Compound C-HuMIGF-1R at a total cumulative radiochemical dose of 0.2 μCi in the following fractions: 0.2 μCi (single dose); 0.1 μCi—twice (2 weeks apart); 0.05 μCi—four times (weekly). Each dose contained approximately 3.2 μg of antibody protein/dose such that the groups received a cumulative protein dose of 3.2, 6.4, or 9.6 μg over one, two or four fractions, respectively. Regression of Colo-205 tumor growth was observed for all dosing regimens out to 85 days from the start of dosing demonstrating that efficacy was dependent on the total radioactivity dose administered rather than the mode of dosing (FIG. 21).

In a second study mice bearing radioresistant lung (A549) tumor xenografts were administered [²²⁵Ac]-Compound C-HuMIGF-1R at a total cumulative radioactivity dose of 0.4 μCi in the following fractions: 0.4 μCi; single dose; 0.2 μCi—twice (2 weeks apart); 0.1 μCi—four times (weekly). Each dose contained approximately 4.1 μg of protein/dose such that the groups received a cumulative protein dose of 4.1, 8.2, or 16.4 μg over one, two or four fractions, respectively. Suppression of A549 tumor growth was observed for all dosing regimens out to 66 days from the start of dosing demonstrating that efficacy was dependent on the total radioactivity dose administered rather than the mode of dosing (FIG. 22). In addition, these data demonstrate targeted alpha-therapy can be effective against cell lines (eg. A549) resistant to ionizing radiation. Furthermore, these data demonstrate that during multiple dosing regimens, injected antibody does not appear to prevent access of subsequent doses to the tumor cells, otherwise the fractionated dosing strategy would be expected to provide reduced efficacy over single a dose in these studies.

Other Embodiments

While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure that come within known or customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth. 

What is claimed is:
 1. A compound comprising a structure:

wherein B is an antibody and the antibody is AVE1642 (SEQ ID NO: 9) or a pharmaceutically acceptable salt thereof.
 2. The compound of claim 1, wherein the compound is in a pharmaceutically acceptable carrier.
 3. The compound of claim 1, wherein said compound is present in a composition that further comprises a metal salt.
 4. The compound of claim 3, wherein the metal salt comprises a radionuclide.
 5. The compound of claim 4, wherein the radionuclide is ¹¹¹In.
 6. The compound of claim 4, wherein the radionuclide is ²²⁵Ac.
 7. A method of treating cancer, the method comprising administering to a subject in need thereof an effective amount of a composition comprising a compound of claim 1 in a pharmaceutically acceptable carrier.
 8. The method of claim 7 wherein the cancer is a solid tumor or hematologic cancer.
 9. The method of claim 7, wherein the cancer is breast cancer, non-small cell lung cancer, small cell lung cancer, pancreatic cancer, head and neck cancer, prostate cancer, colorectal cancer, thyroid cancer, sarcoma, adrenocortical carcinoma, Ewing's Sarcoma, glioblastoma multiforme, liver cancer, neuroendocrine tumors, bladder cancer, gastric and gastroesophageal junction cancers, melanoma, multiple myeloma, or acute myeloid leukemia.
 10. The method of claim 7, further comprising administering an antiproliferative agent, radiation sensitizer, an immunoregulatory, or immunomodulatory agent.
 11. A method of identifying cancer cells expressing IGF-1R, the method comprising administering to a subject an effective amount of a composition comprising a compound of claim 3 to said cells and determining the binding of said composition to said cancer cells, said binding indicating that said cancer cells are cancer cells expressing IGF-1R.
 12. The method of claim 11, wherein the cancer cells are breast cancer cells, non-small cell lung cancer cells, small cell lung cancer cells, pancreatic cancer cells, head and neck cancer cells, prostate cancer cells, colorectal cancer cells, thyroid cancer cells, sarcoma cells, adrenocortical carcinoma cells, Ewing's Sarcoma cells, glioblastoma multiforme cells, liver cancer cells, neuroendocrine tumor cells, bladder cancer cells, gastric and gastroesophageal junction cancer cells, melanoma cells, multiple myeloma cells, or acute myeloid leukemia cells.
 13. The method of claim 11, wherein said metal salt comprises a radionuclide.
 14. The method of claim 11, wherein said compound is in a pharmaceutically acceptable carrier.
 15. The method of claim 7, wherein said compound is present in a composition that further comprises a metal salt.
 16. The method of claim 15, wherein the metal salt comprises a radionuclide.
 17. The method of claim 16, wherein the radionuclide is ²²⁵Ac.
 18. The method of claim 13 wherein said radionuclide is ¹¹¹In. 